Subunit selective NMDA receptor potentiators for the treatment of neurological conditions

ABSTRACT

Provided are compounds, pharmaceutical compositions and methods of treating or preventing disorders associated with NMDA receptor activity, including schizophrenia, Parkinson&#39;s disease, cognitive disorders, depression, neuropathic pain, stroke, traumatic brain injury, epilepsy, and related neurologic events or neurodegeneration. Compounds of the general Formulas A-J, and pharmaceutically acceptable salts, esters, prodrugs or derivatives thereof are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/146,201 filed Sep. 11, 2012, which is a national stage entry of PCTApplication Number PCT/US2010/022439 filed Jan. 28, 2010, which claimsthe benefit of priority to U.S. Provisional Application No. 61/147,897filed Jan. 28, 2009, all hereby incorporated by reference in theirentireties.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grants NS036654and NS065371 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is in the area of NMDA receptor potentiators thatcan be used to treat a wide range of neurological diseases andconditions, and includes methods and compositions for the treatment ofneurological disorders involving NMDA-receptors.

BACKGROUND OF THE INVENTION

The glutamate receptor gene family encodes ligand-gated ion channelsthat can be divided into three classes ((AMPA(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), kainate, andNMDA (N-methyl-D-aspartic acid)) on the basis of agonist pharmacologyand molecular structure (Dingledine et al. 1999; Qian & Johnson 2002;Erreger et al 2004; Wollmuth & Sobolevsky 2004). NMDA receptors mediatea slow, Ca²⁺-permeable component of excitatory synaptic transmission inthe central nervous system, and have garnered considerable attentionbecause of their prominent role in many normal brain functions,including synaptic plasticity (Lisman 2003; Miyamoto 2006), frequencyencoding of information (Froemke et al 2005; Kampa et al 2006; Rhodes2006), and neuronal development (Rudhard et al 2003; Colonnese et al2005, 2006; Waters & Machaalani 2005; Nacher & McEwen 2006). Inaddition, NMDA receptors play an overt role in neuropathology ofischemia and traumatic brain injury (Whetsell 1996; Miyabe et al 1997;Dirnagl et al 1999; Brauner-Osborne et al 2000; Wang & Shuaib 2005).NMDA receptors have been suggested to be involved in a wide range ofneurological diseases, including schizophrenia, depression, psychosis,Huntington's disease, Alzheimer's disease, and Parkinson's disease.

NMDA receptors are tetrameric complexes comprised of glycine-binding NR1subunits, glutamate-binding NR2 subunits, and NR3 (A and B) subunits.The subunit composition determines the functional properties of nativeNMDA receptors.

Expression of the NR1 subunit alone does not produce a functionalreceptor. Co-expression of one or more NR2 subunits or one or more NR3subunits is required to form functional channels. In addition toglutamate, the NMDA receptor requires the binding of a co-agonist,glycine, to allow the receptor to function. A glycine binding site isfound on the NR1 and NR3 subunits, whereas the glutamate binding site isfound on NR2 subunits. The four NR2 subunits (NR2A, B, C, and D) eachendow the receptor with surprisingly divergent single channelconductances, deactivation time courses, and open probabilities (Sternet al 1992; Wyllie et al 1998; Vicini et al 1998; Erreger et al 2004;Erreger et al 2005ab). The increasingly precise anatomical localizationof the NR2 subunits (Akazawa et al 1994; Monyer et al 1994; Buller et al1994; Paquet et al 1997; Dunah et al 1998; Thompson et al 2002; Lau etal 2003; Lopez de Armentia & Sah 2003; Dunah & Standaert 2003; Dunah etal 2003; Hallett & Standaert 2004; Salter & Fern 2005; Karodottir et al2005) has strengthened the therapeutic rationale for the development ofsubunit-selective NMDA receptor potentiators, which should target NMDAreceptor functions in specific brain regions without engaging NMDAreceptors elsewhere. This idea has fueled optimism that NR2subunit-selective modulators might be well-tolerated therapeutic agentsfor a wide variety of different indications.

At resting membrane potentials, NMDA receptors are largely inactive dueto a voltage-dependent block of the channel pore by magnesium ions.Depolarization releases this channel block and permits passage ofcalcium as well as other cations. NR2A- and NR2B-containing NMDAreceptors are more sensitive to Mg²⁺ blockade than NR2C- andNR2D-containing receptors. The NMDA receptor is modulated by a number ofendogenous and exogenous compounds, including, sodium, potassium, andcalcium ions that can not only pass through the NMDA receptor channelbut also modulate the activity of receptors. Zinc blocks the channelthrough NR2A- and NR2B-containing receptors in a noncompetitive andvoltage-independent manner. Polyamines can also either potentiate orinhibit glutamate-mediated responses (See, for example, McGurk et al.,Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 9971-9974, December 1990).

NR2-Subunit Selectivity of Existing NMDA Receptor Modulators

Few NMDA receptor potentiators (or positive modulators) have beendescribed to date in the literature. Perhaps the best known potentiatorsare naturally occurring polyamines and neurosteroids. Extracellularpolyamines such as spermine and spermidine can potentiate with lowpotency (EC₅₀ 100's μM) the function only of NR2B-containing NMDAreceptors (Williams et al 1994; Traynelis et al 1995). In addition,various neurosteroids both potentiate and inhibit NR2A/B receptors,depending on concentration and subunit composition. Neurosteroids bindat much higher rates to closed receptors than active receptors (Horak etal 2004, 2006). It is believed that highly subunit-selective drug-likepotentiators of heterodimeric NMDA receptors containing NR2A, NR2B,NR2C, or NR2D subunits are heretofore unknown. Experience withnon-selective and subunit selective NMDA receptor antagonists suggeststhat subunit selective potentiators will likely have fewer side effectsthan non-selective potentiators that act at all NMDA receptors.Moreover, because subunits show differential distribution in the brain,it stands to reason that identifying compounds that target specificsubunits may bring about a therapeutically useful effect in one brainregion while minimizing effects in brain regions that lack thatparticular subunit.

Clinical Relevance of NMDA Receptor Potentiators: Learning and Memory

Enhancement of NMDA receptor activity has been proposed to be a usefultherapeutic strategy for certain conditions associated with alteredcognitive function (Lisman et al., 2008). Overexpression of the NR2Bsubunit can enhance learning and memory in animal models (Tang et al.,1999, 2001; Cao et al., 2007). In addition, D-cycloserine has beenstudied as an adjunct to behavioral therapy to promote the extinction ofmaladaptive associations. This approach is based on the hypothesis thatD-cycloserine will augment or enhance therapy-directed learning throughpotentiation of NMDA receptor-dependent learning. This potentiation isbelieved to occur through the increased D-serine occupancy of NR1glycine binding sites. D-cycloserine increased the efficacy ofbehavioral therapy in clinical trials involving patients sufferingacrophobia (Ressler et al., 2004), social anxiety disorder (Hofmann etal., 2006), or obsessive compulsive disorder (Kushner et al., 2007;Wilhelm et al., 2008). The complex multi-subunit composition of NMDAreceptors offers further opportunities for pharmacological manipulationfor therapeutic gain while minimizing side effects by allowing therapyto be targeted at brain regions expressing specific NR2 subunits.

Clinical Relevance of NMDA Receptor Potentiators for Schizophrenia,Psychoses, Bipolar Disorder, and Depression

Schizophrenia and psychosis and other neuropsychiatric disorders arisefrom changes in neurotransmitter systems, neuronal connectivity, orboth. The dopamine hyperactivity hypothesis for schizophrenia, supportedby years of clinical experience and neurochemical data, maintains thatoveractivation of dopamine receptors, such as the D2 subtype, leads tocognitive dysfunction that can be treated by competitive dopaminergicantagonists (Hirsch & Barnes 1995; Seeman et al 2006). The recognitionthat NMDA inhibitor-induced behavioral effects closely mimic thesymptoms of schizophrenia (Javitt and Zukin, 1991; Luby et al., 1959)lead to the hypothesis that NMDA receptor hypofunction may be acausative factor in schizophrenia (Javitt, 2007; Krystal et al., 2002;Olney et al., 1999; Tsai and Coyle, 2002; Yamada et al., 2005; Morita etal., 2007). That is, if blockade of NMDA receptors can reproduceschizophrenic symptoms, it stands to reason that hypofunction of theNMDA receptor system might underlie these symptoms (Coyle et al 2003).This hypothesis led to the proposal that potentiation of NMDA receptorfunction may have therapeutic benefit in patients suffering fromschizophrenia (Heresco-Levy, 2000; Morris et al 2005).

Circuit-based models of schizophrenia have further highlighted thepotential role of NMDA receptor hypofunction in interneurons (Lisman etal 2008), which happen to express NR2C and/or NR2D subunits (Monyer etal 1994; Rudolph et al 1996; Thompson et al 2002; Binshtok et al 2006).These models predict that enhancement of interneuron activity (forexample by NR2C/D selective potentiators) could be beneficial forpatients. Similarly, the ability of certain NR2B-selective antagoniststo produce psychosis (Preskorn et al 2008) suggests that potentiation ofNMDA receptors containing the NR2B subunit may also be therapeuticallybeneficial. While not wishing to be bound to a particular theory, suchsubunit-selective potentiators might bind to a site independent of theagonist recognition site, and enhance the proportion of time thereceptor remains open when agonists such as glutamate and glycine bind.Thus, the regional and cell-specific differences in the expression ofthe NR2C and NR2D subunits in interneurons provides a rationale for thedevelopment of NR2C/D-selective potentiators that may improve negativeand cognitive symptoms, or influence mood. Additional support for thisidea is derived from the observation that enhancement of NMDA receptorfunction with glycine-site agonists and glycine transport inhibitors mayimprove negative and cognitive symptoms when used as adjuncts to currentantipsychotic therapies (Javitt et al 1994; Depoortere et al 2005). Thisfinding provided rationale for clinical trials of agonists at theglycine site on the NMDA receptor (Coyle and Tsai, 2004; Labrie andRoder, 2009; Shim et al., 2008). Several studies of the use of glycineand D-serine as adjuncts to antipsychotics therapy revealed moderatereduction of negative symptoms and suggested a trend toward a decreasein cognitive symptoms (Tuominen et al., 2005). One subsequent clinicaltrial suggested a beneficial effect of the glycine site agonistD-alanine on both positive and negative symptoms of schizophrenia (Tsaiet al., 2006). D-cycloserine, an antibiotic and glycine site ligand, isa partial agonist at the glycine site and preferentially activates NMDAreceptors containing the NR2C subunit (Sheinen et al., 2001; Dravid etal., 2010). Initial clinical studies of D-cycloserine indicated abeneficial effect on negative symptoms (Goff et al., 1999). Becausepreclinical data suggest the possibility of tachyphylaxis to glycinesite ligands, D-cycloserine has been examined with intermittent dosing,which improved negative symptoms in patients suffering schizophrenia(Goff et al., 2008a). Thus, there is general optimism that if allostericpotentiators of NMDA receptor function could be found, such compoundsmight provide beneficial effects by reducing NMDA receptor hypofunctionin psychoses and schizophrenia (Heresco-Levy 2005; Lindsley et al 2006),or perhaps by having antidepressant effects.

Additional circumstantial and correlative data are consistent with arole for NR2C/D subunit in schizophrenia, psychoses, andneuropsychiatric conditions. NR2D subunit mRNA is significantlyincreased in the prefrontal cortex of schizophrenic patients (Akbarianet al 1996), and NR2D protein expression increases in the frontal cortexof PCP-treated rats (Lindahl & Keifer 2004). Because NMDA receptorhypofunction in frontal and prefrontal cortex correlates with negativesymptoms and cognitive impairments (Andreasen et al 1997; Molina et al2005), an NR2D potentiator might be a useful therapy to treat thesesymptoms. Interestingly, genetic analysis of polymorphisms suggests thatthe NR2D gene may be a locus contributing to schizophreniasusceptibility in the Japanese population (Makino et al 2005).

Clinical Relevance of NMDA Receptor Potentiators: Facilitation of MotorLearning During Rehabilitation

Of the 1.4 million people who sustain a traumatic brain injury (TBI)each year in the United States, 50,000 die, 235,000 are hospitalized,and 1.1 million are treated and released from an emergency department(Langlois et al., 2006). The Centers for Disease Control and Preventionestimates that at least 5.3 million Americans currently have a long-termor lifelong need for help to perform activities of daily living as aresult of a TBI (Thurman et al 1999). On average, every 45 secondssomeone in the United States has a stroke, giving rise to 700,000 casesof stroke every year, of which 75% are likely to survive with impairedfunction requiring rehabilitation (Jorgensen et al., 1995). Millions ofstroke and TBI survivors thus suffer from a movement-related problem.Together, stroke and TBI have a greater disability impact than virtuallyall other neurological conditions and chronic diseases.

The exceptionally large number of patients suffering from disabilitiescreates a strong need to develop new treatments to facilitate recoveryof cortical function following acute neural insults, as occur inischemic conditions, stroke and traumatic brain injury. Accomplishmentof this task has the potential to improve clinical outcomes and qualityof life for patients suffering brain injury, stroke, hypoxia, orischemia. Several recent studies involving NMDA receptors suggest a pathtowards development of new therapies to enhance cortical motor learningand facilitate recovery from brain insult (See, for example, Nitsche etal., Neuropsychopharmacology (2004) 29, 1573-1578). NMDA receptorsrequire the simultaneous binding of two ligands (glycine, glutamate)before they open to initiate depolarizing current flow into a neuron. Aclinically approved partial agonist at the glycine site of the NMDAreceptor (D-cycloserine) influences emotional learning, being apotentiator of extinction of conditioned fear in both animal models andhuman anxiety disorders (Walker et al., 2002; Ressler et al., 2004;Hofmann et al., 2006ab). If motor learning is amenable topharmacological manipulation in the same manner as emotional learning,this may provide a means to improve rehabilitation of patients sufferingneuronal loss as a consequence of stroke or TBI through enhancement ofNMDA receptor function during physical therapy.

While the molecular basis for the behavioral effects of D-cycloserinehas not been elucidated, several clues exist as to why D-cycloserinemight have unique behavioral actions that other partial or full agonistsat the either the glycine or glutamate binding site on the NMDA receptorappear to lack. NMDA receptors are comprised of NR1 and NR2 subunits,and D-cycloserine at maximally effective concentrations appears to causeslightly lower responses than maximally effective levels of glycine (theendogenous ligand) at NMDA receptors comprised of NR1/NR2A, NR1/NR2B,and NR1/NR2D subunits. By contrast, a recent study demonstrated thatD-cycloserine causes current responses at NR1/NR2C receptors that arenearly twice as large as the endogenous agonist glycine (Sheinin et al.,2002). That is, the agonist D-cycloserine appears to selectively enhanceNMDA receptor function when the NR2C subunit is present through itsbinding to the glycine recognition site on the NR1 subunit. This findingsuggests that the unique behavioral effects of D-cycloserine may berelated to the potentiation of NR2C-containing NMDA receptors. Implicitin this hypothesis is the idea that enhancement of only NMDA receptorsthat contain the NR2C subunit may enhance emotional learning.

In cortical structures (hippocampus and neocortex), NR2C subunit mRNA isexpressed in subsets of interneurons (Monyer et al., 1994; Binshtock etal., 2006), suggesting that modulation of NR2C function has thepotential to sculpt network activity through modulation of interneuronalfiring. Thus, NR2C potentiators may be useful as cognitive enhancers,with many potential functions, including treatment and prevention ofneurodegenerative diseases associated with cognitive decline. Inaddition, subunit selective NMDA receptor potentiation may be useful forimproving rehabilitation, for example, from stroke and traumatic braininjury.

Clinical Relevance of NMDA Receptor Potentiators: Modulation of MotorFunction

NMDA receptors containing the NR2C subunit are highly expressed incerebellum (Monyer et al 1994; Lansola et al., 2005), a structure wellknown to be important for sculpting motor function, in particularcoordination and fine motor movement. NR2C is particularly abundant atthe mossy-fiber-granule cell synapse, and thus modulators of NR2C mayhave effects on cerebellar function through actions at this synapse,which ultimately gives rise to the input to Purkinje cells via theparallel fibers. In addition, NR2D subunits have been proposed to beexpressed by neurons of the deep cerebellar nuclei (Cull-Candy et al.,1998), providing another target for influencing cerebellar function.Thus, NR2C/D-selective NMDA receptor potentiators may controlinformation processing within the cerebellum, and thus have usefuleffects on motor function, coordination, motor learning, or movementcontrol. Therefore, NR2C/D potentiators can be used to treat a widerange of neurological diseases associated with impaired motor function.

Clinical Relevance of NMDA Receptor Potentiators: Epilepsy

Interneurons typically utilize the inhibitory neurotransmitter GABA andcontact a large number of cells. Interneuron firing thus has the abilityto hyperpolarize large numbers of neurons. In this way, interneurons canhave far-reaching effects on neuronal excitability and signal processingin the central nervous system. Epilepsy is a disorder associated withhypersynchronous and excessive neuronal firing, giving rise to bothelectrographic and motor seizures. Interneuron inhibition is thought tolimit excessive tissue excitability, and a number of compounds thatenhance GABA receptor function (e.g. phenobarbital, benzodiazepine) areuseful as anticonvulsant agents in some settings. Because NR2C- andNR2D-containing receptors are expressed in hippocampal and corticalinhibitory interneurons but not excitatory principle cells (Monyer et al1994; Rudolph et al 1996; Thompson et al 2002; Binshtok et al 2006),modulators that selectively enhance NR2C and NR2D receptor functionshould depolarize interneurons, and thereby increase firing of GABAergicinterneurons. As interneurons fire more action potentials, the resultingrelease of GABA onto excitatory principle cells exerts an inhibitoryeffect that can be anticonvulsant. Thus, NR2C and NR2D potentiators canbe used for their anticonvulsant properties.

Treatment of Bone Disorders

NMDA receptors of the NR2D subtype are found in the osteoblasts, andtherefore, compounds which have activity at these receptors can beuseful in treating bone disorders.

The bone-remodeling cycle occurs at particular areas on the surfaces ofbones. Osteoclasts which are formed from appropriate precursor cellswithin bones resorb portions of bone; new bone is then generated byosteoblastic activity. Osteoblasts synthesise the collagenous precursorsof bone matrix and also regulate its mineralization. The dynamicactivity of osteoblasts in the bone remodelling cycle to meet therequirements of skeletal growth and matrix and also regulate itsmaintenance and mechanical function is thought to be influenced byvarious factors, such as hormones, growth factors, physical activity andother stimuli. Osteoblasts are thought to have receptors for parathyroidhormone and estrogen. Ostoeclasts adhere to the surface of boneundergoing resorption and are thought to be activated by some form ofsignal from osteoblasts.

Irregularities in one or more stages of the bone-remodelling cycle (e.g.where the balance between bone formation and resorption is lost) canlead to bone remodelling dirorders, or metabolic bone diseases. Examplesof such diseases are osteoporosis, Paget's disease and rickets. Some ofthese diseases are caused by over-activity of one half of thebone-remodelling cycle compared with the other, i.e. by osteoclasts orosteoblasts. In osteoporosis, for example, there is a relative increasein osteoclastic activity which may cause a reduction in bone density andmass. Osteoporosis is the most common of the metabolic bone diseases andmay be either a primary disease or may be secondary to another diseaseor other diseases.

Post-menopausal osteoporosis is currently the most common form ofosteoporosis. Senile osteoporosis afflicts elderly patients of eithersex and younger individuals occasionally suffer from osteoporosis.

Osteoporosis is characterized generally by a loss of bone density.Thinning and weakening of the bones leads to increased fracturing fromminimal trauma. The most prevalent fracturing in post-menopausalosteoporotics is of the wrist and spine. Senile osteoporosis, ischaracterized by a higher than average fracturing of the femur.

The tight coupling between the osteoblastic and osteoclastic activitiesof the bone remodeling cycle make the replacement of bone already lostan extremely difficult challenge. Consequently, research into treatmentsfor prevention or prophylaxis of osteoporosis (as opposed to replacementof already-lost bone) has yielded greater results to date.

Estrogen deficiency has been considered to be a major cause ofpost-menopausal osteoporosis. Indeed steroids including estrogen havebeen used as therapeutic agents (New Eng. J. Med., 303, 1195 (1980)).However, recent studies have concluded that other causes must exist (J.Clin. Invest., 77, 1487 (1986)).

Other bone diseases can be caused by an irregularity in thebone-remodeling cycle whereby both increased bone resorption andincreased bone formation occur. Paget's disease is one such example.

There remains a need for improved neuroprotective compounds and methodsfor the treatment of neuropathologies that have reduced toxicity. Thereis also a need for improved treatments for neuropathic pain,inflammatory pain, stroke, traumatic brain injury, global ischemia,hypoxia, spinal cord trauma, epilepsy, addiction, depression,schizophrenia, motor disorders, and neurodegenerative diseases anddisorders.

It would be advantageous to have compounds, compositions including thecompounds, and methods of treatment using the compounds to treat thesedisorders. The present invention provides such compounds, compositions,and methods of treatment.

SUMMARY OF THE INVENTION

NMDA receptor potentiators, including NMDA receptor potentiators ofFormulas A-J, and pharmaceutically acceptable salts, esters, prodrugsand derivatives thereof, are provided. Also provided are compositionsand methods of using these compounds to treat or prevent a variety ofneurological disorders, to provide neuroprotection, to preventneurodegeneration, to treat neuropathic pain, to control addiction, toease the symptoms of drug withdrawal, to improve cognition, and to treatschizophrenia, psychoses, depression, and the like. The compounds can beused to treat motor disorders, including tardive diskinesia, to treatbipolar and other neuropsychiatric disorders, including anxiety anddepression, and can provide cognitive enhancement. The compounds can beused in patients with normal NMDA receptor expression, so long as thedisorder involves the NMDA receptors, and the disorders are not limitedto those involving neurodegeneration.

In certain embodiments, the compounds are used for treatingschizophrenia, depression, bipolar disorder, obsessive compulsivedisorder, neuropathic and inflammatory pain, stroke, traumatic braininjury, epilepsy, other neurologic events or neurodegeneration whoseonset or subsequent effects involve NMDA receptor activation,Parkinson's disease, Alzheimer's disease, Huntington's chorea, ALS, andother neurodegenerative conditions known in the art or predicted to beresponsive to treatment using NMDA receptor potentiators. In particularembodiments, the compounds are used for the prophylaxis ofschizophrenia, depression, neuropathic or inflammatory pain, stroke,traumatic brain injury, epilepsy, other neurologic events orneurodegeneration resulting from NMDA receptor activation, Parkinson'sdisease, Alzheimer's disease, Huntington's chorea, ALS, and otherneurodegenerative conditions. The compounds can be administered on aprophylactic basis to a patient at risk of a disorder associated withNMDA receptor hypofunction. In particular embodiments, the compounds canact as neuroprotective agents by acting on multiple neurons to alter theoverall balance of circuit activity.

Osteoblasts have a relatively high concentration of NMDA 2D receptorsubtype, but not other receptor subtypes. The compounds described hereinwhich are specific for the NMDA 2D receptor subtype can be used to treatbone disorders, by turning on or turning off bone formation. Thecompounds described herein can thus enhance bone formation and bonedensity and have beneficial effects on the activity and differentiationof bone cells.

Accordingly, in one embodiment, the present invention relates to amethod for enhancing bone formation in a mammal in need thereof, such asa human, comprising administering to the mammal an effective amount of acompound described herein that is specific for the NMDA 2D receptorsubtype. The mammal may have a bone deficit or be at risk of developinga bone deficit, or have a bone remodeling disorder or is at risk ofdeveloping such disorder. Examples of bone remodeling disorders includeosteoporosis, Paget's disease, osteoarthritis, rheumatoid arthritis,achondroplasia, osteochodrytis, hyperparathyroidism, osteogenesisimperfecta, congenital hypophosphatasia, fribromatous lesions, fibrousdisplasia, multiple myeloma, abnormal bone turnover, osteolytic bonedisease and periodontal disease. In one aspect of this embodiment, thebone remodeling disorder is osteoporosis, including primaryosteoporosis, secondary osteoporosis, post-menopausal osteoporosis, maleosteoporosis and steroid-induced osteoporosis.

The compounds can also be used to enhance bone formation in a mammalhaving a bone deficit which does not result from a bone remodelingdisorder. Such bone deficits may result, for example, from a bonefracture, bone trauma, or a condition associated with post-traumaticbone surgery, post-prosthetic joint surgery, post-plastic bone surgery,post-dental surgery, bone chemotherapy treatment or bone radiotherapytreatment. The present invention also provides a method for increasingbone density, stimulating osteoblast differentiation, inhibitingosteoclast differentiation, activating the bone formation activity ofdifferentiated osteoblasts, simultaneously stimulating osteoblastdifferentiation and inhibiting osteoclast differentiation.

The compounds can be administered in combination with at least one boneenhancing agent. Examples of suitable bone enhancing agents include asynthetic hormone, a natural hormone, oestrogen, calcitonin, tamoxifen,a bisphosphonate, a bisphosphonate analog, vitamin D, a vitamin Danalog, a mineral supplement, a statin drug, a selective oestrogenreceptor modulator and sodium fluoride.

The compounds can be administered alone, or in combination oralternation with other compounds useful for treating or preventing otherneurologic events or neurodegeneration resulting from NMDA receptoractivation, Parkinson's disease, Alzheimer's disease, Huntington'schorea, ALS, and other neurodegenerative or neurological conditionsknown to the art to be responsive to treatment using NMDA receptorpotentiators

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chart showing time course of fluorescence responses of theNR1/NR2D cell line; data points are displayed as F/F_(BASELINE), where Fis the fluorescence measured after addition of glutamate/glycine,buffer, or 1 μM MK801 to the well. Data points are mean±SD of 4-5 wells.Final agonist concentrations were 100 μM glutamate/1 mM glycine in thepresence of 30 μM of the competitive glycine antagonist 7-Cl-kynurenate.

FIG. 2A shows two electrode voltage clamp recordings of recombinantNMDA, AMPA, and kainate receptors activated by maximally effectiveconcentrations of glutamate and glycine (100, 50 μM). Values aremean±SEM (n=14-20). FIG. 2B shows how a specific dihydroisoquinolinecompound, herein referred to as DIQ-1180, which is a potentiator ofNR2C/D-containing NMDA receptors, increased the opening frequency ofrecombinant NR1/NR2D single channel currents in outside-out patchesactivated by a maximally effective concentration of glutamate/glycine. Cis the closed level; broken line is the open level.

FIG. 3 is a photomicrograph of recording arrangement for the subthalamicnucleus. All neurons had an I_(H) current, as expected for subthalamicneurons. Responses to pressure-applied NMDA/glycine (both 2 mM, 20 ms, 2psi) in slices bathed in 0.2 mM Mg²+, 0.5 μM TTX, 10 μM bicuculline, 10μM CNQX were potentiated by 30 μM DIQ-1180, and blocked by 100 μMDL-APV.

FIG. 4A is a chart showing the construct design for the NR2D-expressingBHK cell line used in Example 8. FIG. 4B is a series of photographsshowing that induction of NR1 was visualized using the monoclonal mAb54.1. The control is black, as there is no fluorescence. The 24 hourinduction by DOX produces spots, which in a color photograph wouldappear as green dots indicating immunofluorescence. FIG. 4C is a seriesof photographs showing Fura-2 based imaging of a BHK cell lineexpressing NR1/NR2D during challenge with 100 μM glutamate plus 30 μMglycine. Ratio images are shown for 340/380 nm excitation (510 nmemission) for Fura-2 before and after challenge with glutamate plusglycine.

DETAILED DESCRIPTION

It has been discovered that certain NMDA receptor potentiators areuseful for treating or preventing a wide variety of central nervoussystem disorders. The potentiators, pharmaceutical compositionsincluding the potentiators, methods for their synthesis, and methods oftreatment using the potentiators, are described in detail below.

The present invention will be better understood with reference to thefollowing definitions:

Definitions

Whenever a term in the specification is identified as a range (i.e. C₁₋₄alkyl), the range independently refers to each element of the range. Asa non-limiting example, C₁₋₄ alkyl means, independently, C₁, C₂, C₃ orC₄ alkyl. Similarly, when one or more substituents are referred to asbeing “independently selected from” a group, this means that eachsubstituent can be any element of that group, and any combination ofthese groups can be separated from the group. For example, if R¹ and R²can be independently selected from X, Y and Z, this separately includesthe groups R¹ is X and R² is X; R¹ is X and R² is Y; R¹ is X and R² isZ; R¹ is Y and R² is X; R¹ is Y and R² is Y; R¹ is Y and R² is Z; R¹ isZ and R² is X; R¹ is Z and R² is Y; and R¹ is Z and R² is Z.

The term “NMDA receptor” as used herein means a postsynaptic receptorwhich is stimulated, at a minimum, by the excitatory amino acidsglutamate and glycine. It is a ligand-gated receptor with astrychnine-insensitive glycine site.

The term “potentiator” as used herein means any compound that increasesthe flow of current through the NMDA receptor when the agonists requiredfor activity are bound to the receptor. That is, a potentiator enhancesthe response of NMDA receptors by binding to a site other than theagonist binding site.

The term “co-agonist” as used herein means any pair of compounds (forexample glutamate and glycine) that bind to the same receptor complex atdifferent sites and for which binding by both molecules is required toactivate the receptor.

The term “antagonist” as used herein means any compound which reducesthe flow of current through the NMDA receptor either by blocking thebinding of an agonist or co-agonist, blocking the channel, causingchannel closure, or binding to a site separate from the agonist bindingsites and channel pore that, when occupied, inhibits receptor function.

The term “ligand” as used herein means any compound which binds to asite on the NMDA receptor.

The term “halogen” or “halo” as used herein refers to fluorine,chlorine, bromine, and iodine atoms.

The term “hydroxyl” as used herein means C—OH.

The term “lower alkoxy” as used herein means lower alkyl-O—.

The term “oxo” as used herein means a C═O group.

The term “mercapto” as used herein means a C—SH group.

The term “aryl” as used herein means an organic radical derived from anaromatic hydrocarbon, e.g., phenyl from benzene.

The term “amino” as used herein means —NH₂.

The term “alkyl” is used herein, unless otherwise specified, refers to asubstituted or unsubstituted, saturated, straight, branched, or cyclic(also identified as cycloalkyl), primary, secondary, or tertiaryhydrocarbon, including but not limited to those of C1 to C6.Illustrative examples of alkyl groups are methyl, ethyl, propyl,isopropyl, cyclopropyl, butyl, sec-butyl, isobutyl, tert-butyl,cyclobutyl, 1-methylbutyl, 1,1-dimethylpropyl, pentyl, cyclopentyl,isopentyl, neopentyl, cyclopentyl, hexyl, isohexyl, and cyclohexyl.Unless otherwise specified, the alkyl group can be unsubstituted orsubstituted with one or more moieties selected from the group consistingof alkyl, halo, haloalkyl, hydroxyl, carboxyl, acyl, acyloxy, amino,amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino,alkoxy, aryloxy, nitro, cyano, thio, sulfonyl, ester, carboxylic acid,amide, phosphonyl, phosphinyl, thioether, oxime, or any other viablefunctional group that does not inhibit the pharmacological activity ofthis compound, either unprotected, or protected as necessary, as knownto those skilled in the art, for example, as taught in Greene, et al.,Protective Groups in Organic Synthesis, John Wiley and Sons, ThirdEdition, 2002. In certain embodiments, alkyl may be optionallysubstituted by one or more fluoro, chloro, bromo, iodo, hydroxy,heterocyclic, heteroaryl, carboxy, alkoxy, nitro, NH₂, N(alkyl)₂,NH(alkyl), alkoxycarbonyl, —N(H or alkyl)C(O)(H or alkyl), —N(H oralkyl)C(O)N(H or alkyl)₂, —N(H or alkyl)C(O)O(H or alkyl), —OC(O)N(H oralkyl)₂, —S(O)_(n)—(H or alkyl), —C(O)—N(H or alkyl)₂, cyano, alkenyl,cycloalkyl, acyl, hydroxyalkyl, heterocyclic, heteroaryl, aryl,aminoalkyl, oxo, carboxyalkyl, —C(O)—NH₂, —C(O)—N(H)O(H or alkyl),—S(O)₂—NH₂, —S(O)_(n)—N(H or alkyl)₂ and/or —S(O)₂—N(H or alkyl)₂, wheren in this instance is 1 or 2.

The term “lower alkyl” as used herein means an alkyl radical having 1-9carbon atoms, which may be straight or branched, including, for example,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, amyl,isoamyl, hexyl, heptyl, octyl, nonyl, or the like.

The term “cycloalkyl” is used herein, unless otherwise specified, refersto a substituted or unsubstituted, saturated cyclic hydrocarbon,including but not limited to those of C₃ to C₁₂. Illustrative examplesof cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl. Unless otherwise specified, the cycloalkyl group can beunsubstituted or substituted with one or more moieties selected from thegroup consisting of alkyl, halo, haloalkyl, hydroxyl, carboxyl, acyl,acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, thio, sulfonyl, ester,carboxylic acid, amide, phosphonyl, phosphinyl, thioether, oxime, or anyother viable functional group that does not inhibit the pharmacologicalactivity of this compound, either unprotected, or protected asnecessary, as known to those skilled in the art, for example, as taughtin Greene, et al., Protective Groups in Organic Synthesis, John Wileyand Sons, Third Edition, 2002. In certain embodiments, the cycloalkylmay be optionally substituted by one or more fluoro, chloro, bromo,iodo, hydroxy, heterocyclic, heteroaryl, carboxy, alkoxy, nitro, NH₂,N(alkyl)₂, NH(alkyl), alkoxycarbonyl, —N(H or alkyl)C(O)(H or alkyl),—N(H or alkyl)C(O)N(H or alkyl)₂, —N(H or alkyl)C(O)O(H or alkyl),—OC(O)N(H or alkyl)₂, —S(O)_(n)—(H or alkyl), —C(O)—N(H or alkyl)₂,cyano, alkenyl, cycloalkyl, acyl, hydroxyalkyl, heterocyclic,heteroaryl, aryl, aminoalkyl, oxo, carboxyalkyl, —C(O)—NH₂,—C(O)—N(H)O(H or alkyl), —S(O)₂—NH₂, —S(O)_(n)—N(H or alkyl)₂ and/or—S(O)₂—N(H or alkyl)₂.

The term “heterocyclic” refers to a non-aromatic or aromatic cyclicgroup wherein there is at least one heteroatom, such as oxygen, sulfur,nitrogen, or phosphorus in the ring. The term “heteroaryl” or“heteroaromatic,” refers to an aromatic that includes at least onesulfur, oxygen, nitrogen or phosphorus in the aromatic ring. Nonlimitingexamples of heteroaryl and heterocyclic groups include furyl, furanyl,pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl,pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl,benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl,benzimidazolyl, purinyl, carbazolyl, oxazolyl, thiazolyl, isothiazolyl,1,2,4-thiadiazolyl, pyrrolyl, quinazolinyl, cinnolinyl, phthalazinyl,xanthinyl, hypoxanthinyl, thiophene, furan, pyrrole, isopyrrole,pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, oxazole,isoxazole, thiazole, isothiazole, pyrimidine or pyridazine, pteridinyl,aziridines, thiazole, isothiazole, oxadiazole, thiazine, pyridine,pyrazine, piperazine, piperidine, pyrrolidine, oxaziranes, phenazine,phenothiazine, morpholinyl, pyrazolyl, pyridazinyl, pyrazinyl,quinoxalinyl, xanthinyl, hypoxanthinyl, pteridinyl, 5-azacytidinyl,5-azauracilyl, triazolopyridinyl, imidazolopyridinyl,pyrrolopyrimidinyl, pyrazolopyrimidinyl, adenine, N6-alkylpurines,N6-benzylpurine, N6-halopurine, N6-vinypurine, N6-acetylenic purine,N6-acyl purine, N6-hydroxyalkyl purine, N6-thioalkyl purine, thymine,cytosine, 6-azapyrimidine, 2-mercaptopyrimidine, uracil,N5-alkylpyrimidines, N5-benzylpyrimidines, N5-halopyrimidines,N5-vinylpyrimidine, N5-acetylenic pyrimidine, N5-acyl pyrimidine,N5-hydroxyalkyl purine, N6-thioalkyl purine, and isoxazolyl. Theheteroaromatic or heterocyclic group can be optionally substituted withone or more substituents selected from halogen, haloalkyl, alkyl,alkoxy, hydroxy, carboxyl derivatives, amido, amino, alkylamino,dialkylamino. The heteroaromatic can be partially or totallyhydrogenated as desired. Nonlimiting examples include dihydropyridineand tetrahydrobenzimidazole. In some embodiment, the heteroaryl may beoptionally substituted by one or more fluoro, chloro, bromo, iodo,hydroxy, thiol, ether, thioether, heterocyclic, heteroaryl, carboxy,alkoxy, nitro, NH₂, N(alkyl)₂, NH(alkyl), alkoxycarbonyl, —N(H oralkyl)C(O)(H or alkyl), —N(H or alkyl)C(O)N(H or alkyl)₂, —N(H oralkyl)C(O)O(H or alkyl), —OC(O)N(H or alkyl)₂, —S(O)_(n)—(H or alkyl),—C(O)—N(H or alkyl)₂, cyano, alkenyl, cycloalkyl, acyl, hydroxyalkyl,heterocyclic, heteroaryl, aryl, aminoalkyl, oxo, carboxyalkyl,—C(O)—NH₂, —C(O)—N(H)O(H or alkyl), —S(O)₂—NH₂, —S(O)_(n)—N(H or alkyl)₂and/or —S(O)₂—N(H or alkyl)₂. Functional oxygen and nitrogen groups onthe heteroaryl group can be protected as necessary or desired. Suitableprotecting groups are well known to those skilled in the art, andinclude trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, andt-butyldiphenylsilyl, trityl or substituted trityl, alkyl groups, acylgroups such as acetyl and propionyl, methanesulfonyl, andp-tolylsulfonyl.

The term “aryl,” unless otherwise specified, refers to a carbon basedaromatic ring, including phenyl, biphenyl, or naphthyl. The aryl groupcan be optionally substituted with one or more moieties selected fromthe group consisting of hydroxyl, acyl, amino, halo, alkylamino, alkoxy,aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,phosphate, or phosphonate, either unprotected, or protected asnecessary, as known to those skilled in the art, for example, as taughtin Greene, et al. Protective Groups in Organic Synthesis, John Wiley andSons, Third Edition, 2002. In certain embodiments, the aryl group isoptionally substituted by one or more fluoro, chloro, bromo, iodo,hydroxy, heterocyclic, heteroaryl, carboxy, alkoxy, nitro, NH₂,N(alkyl)₂, NH(alkyl), alkoxycarbonyl, —N(H or alkyl)C(O)(H or alkyl),—N(H or alkyl)C(O)N(H or alkyl)₂, —N(H or alkyl)C(O)O(H or alkyl),—OC(O)N(H or alkyl)₂, —S(O)_(n)—(H or alkyl), —C(O)—N(H or alkyl)₂,cyano, alkenyl, cycloalkyl, acyl, hydroxyalkyl, heterocyclic,heteroaryl, aryl, aminoalkyl, oxo, carboxyalkyl, —C(O)—NH₂,—C(O)—N(H)O(H or alkyl), —S(O)₂—NH₂, —S(O)_(n)—N(H or alkyl)₂ and/or—S(O)₂—N(H or alkyl)₂.

The term “aralkyl,” unless otherwise specified, refers to an aryl groupas defined above linked to the molecule through an alkyl group asdefined above. The term “alkaryl,” unless otherwise specified, refers toan alkyl group as defined above linked to the molecule through an arylgroup as defined above. Other groups, such as acyloxyalkyl, alkoxyalkyl,alkoxycarbonyl, alkoxycarbonylalkyl, alkylaminoalkyl, alkylthioalkyl,amidoalkyl, aminoalkyl, carboxyalkyl, dialkylaminoalkyl, haloalkyl,heteroaralkyl, heterocyclic-alkyl, hydroxyalkyl, sulfonamidoalkyl,sulfonylalkyl and thioalkyl are named in a similar manner.

The term “alkoxy” or “aryloxy” unless otherwise specified, refers to amoiety of the structure —OR¹, where R¹ is (although defined elsewhere asincluding H), an alkyl, aryl, alkaryl or aralkyl group, or substitutedalkyl, aryl, aralkyl or alkaryl group, as such groups are definedherein.

The term “acyl,” refers to a group of the formula C(O)R′ or “alkyl-oxy”,wherein R′ is an alkyl, aryl, alkaryl or aralkyl group, or substitutedalkyl, aryl, aralkyl or alkaryl.

The term “alkenyl” The term “alkenyl” means a monovalent, unbranched orbranched hydrocarbon chain having one or more double bonds therein. Thedouble bond of an alkenyl group can be unconjugated or conjugated toanother unsaturated group. Suitable alkenyl groups include, but are notlimited to (C₂-C₈)alkenyl groups, such as vinyl, allyl, butenyl,pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl,2-ethylhexenyl,2-propyl-2-butenyl,4-(2-methyl-3-butene)-pentenyl. Analkenyl group can be unsubstituted or substituted with one or twosuitable substituents.

The term “carbonyl” refers to a functional group composed of a carbonatom double-bonded to an oxygen atom: —C═O. Similarly, C(O) or C(═O)refers to a carbonyl group.

The term “amino” refers to —NH₂, —NH(alkyl) or —N(alkyl)₂.

The term “thio” indicates the presence of a sulfur group. The prefixthio- denotes that there is at least one extra sulfur atom added to thechemical. The prefix ‘thio-’ can also be placed before the name of acompound to mean that an oxygen atom in the compound has been replacedby a sulfur atom. The terms ‘thio’ and ‘thiol’ are used interchangeably,unless otherwise indicated.

The term “amido” indicates a group (H or alkyl)-C(O)—NH—.

The term “carboxy” designates the terminal group —C(O)OH.

The term “sulfonyl” indicates an organic radical of the general formula(H or alkyl)-S(═O)₂—(H or alkyl’), where there are two double bondsbetween the sulfur and oxygen.

The term “pharmaceutically acceptable salt” refers to salts or complexesthat retain the desired biological activity of the compounds of thepresent invention and exhibit minimal undesired toxicological effects.Nonlimiting examples of such salts are (a) acid addition salts formedwith inorganic acids (for example, hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid, and the like), and saltsformed with organic acids such as acetic acid, oxalic acid, tartaricacid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannicacid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, naphthalenedisulfonic acid, and polygalacturonic acid; (b) baseaddition salts formed with metal cations such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium,potassium, and the like, or with a cation formed from ammonia,N,N-dibenzylethylenediamine, D-glucosamine, tetraethylammonium, orethylenediamine; or (c) combinations of (a) and (b); e.g., a zinctannate salt or the like. Also included in this definition arepharmaceutically acceptable quaternary salts known by those skilled inthe art, which specifically include the quaternary ammonium salt of theformula —NR⁺A⁻, wherein R is H or alkyl and A is a counterion, includingchloride, bromide, iodide, —O-alkyl, toluenesulfonate, methylsulfonate,sulfonate, phosphate, or carboxylate (such as benzoate, succinate,acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate,benzoate, cinnamoate, mandeloate, benzyloate, and diphenylacetate).

The term “protected” as used herein and unless otherwise defined refersto a group that is added to an oxygen, nitrogen, or phosphorus atom, oron an acetylenic carbon (i.e., a trimethylsilyl group in place of anacetylenic proton) to prevent its further reaction or for otherpurposes. A wide variety of oxygen, nitrogen, phosphorus, and acetylenicprotecting groups are known to those skilled in the art of organicsynthesis, and described in Greene and Wuts, Protective Groups inOrganic Synthesis, supra.

It should be understood that the various possible stereoisomers of thegroups mentioned above and herein are within the meaning of theindividual terms and examples, unless otherwise specified.

As an illustrative example, “1-methyl-butyl” exists in both (R) and the(S) form, thus, both (R)-1-methyl-butyl and (S)-1-methyl-butyl iscovered by the term “1-methyl-butyl”, unless otherwise specified.

I. Compounds

The compounds typically have one of the following Formulas A-J, as shownbelow. The compounds typically have EC₅₀ values in the range of 0.01 to10 μM, 0.01 to 9 μM, 0.01 to 8 μM, 0.01 to 7 μM, 0.01 to 6 μM, 0.01 to 5μM, 0.01 to 4 μM, 0.01 to 3 μM, 0.01 to 2 μM, 0.01 to 1 μM, 0.05 to 7μM, 0.05 to 6 μM, 0.05 to 5 μM, 0.05 to 4 μM, 0.05 to 3 μM, 0.05 to 2μM, 0.05 to 1 μM, 0.05 to 0.5 μM, 0.1 to 7 μM, 0.1 to 6 μM, 0.1 to 5 μM,0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1 μM, 0.1 to 0.5 μM, 0.1to 0.4 μM, 0.1 to 0.3 μM, or 0.1 to 0.2 μM.

Formulas A and B are provided below:

wherein for Formulas A and B,

-   -   Ar₃=

-   -   Ar₄=

X is, independently, N or C bonded to H or a substituent, J, with theproviso that no more than three of X are N;

Y is independently selected from O, S, NR¹, CH₂, and CR¹ ₂;

R¹ and R² are, independently, selected from H, alkyl, substituted alkyl,alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, and hydroxy, and, when R¹ is attached to acarbon atom, it can be halo or cyano,

T is, independently, CHR¹, CR¹ ₂, O, S, or NR¹,

V is, independently, N, or C bonded to H or a substituent J,

J is a non-hydrogen substituent selected from the group consisting ofhalo (—F, —Cl, —Br, —I), nitro, amino (NR¹R²), OR¹, SR¹, —R¹, —CF₃, —CN,—C₂R¹, —SO₂CH₃, —C(═O)NR¹R²—NR′C(═O)R¹, —C(═O)R¹, —C(═O)OR¹,—(CH₂)_(q)OR¹, —OC(═O)R¹, —OC(═O)NR¹R², —NR¹(C═Y)—NR¹R², —NR¹(C═Y)—OH,—NR¹(C═Y)—SH, sulfonyl, sulfinyl, phosphoryl, and azo, and

q is 0-5.

In one embodiment, the aryl ring moiety Ar₄ is replaced with a saturatedring.

Representative structures within Formula A and B (also referred toherein as the 1180 class of compounds) include:

wherein J, T, V, X, and Y are defined above, and n, o, p, and q, arefrom 0-3.

Other representative structures falling within the scope of Formulas Aand B are as follows:

wherein

X is, independently, N or C bonded to H or a substituent, J, with theproviso that no more than three of X are N;

T is, independently, C(R¹)₂, NR¹, O or S,

V is, independently, N, or C bonded to H or a substituent J,

Y is selected from O, S, NR¹, CH₂, and CR¹ ₂;

R¹ and R² are independently selected from H, alkyl, substituted alkyl,alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, and hydroxy,

R¹ and R² can optionally join to form a C₃₋₁₀ heterocyclic moiety, whichheterocyclic moiety can optionally include a second heteroatom selectedfrom O, S, and N,

z is an integer from 0 to 3

and

J is a non-hydrogen substituent selected from the group consisting ofhalo (—F, —Cl, —Br, —I), nitro, amino (NR¹R²), OR¹, SR¹, —R¹, —CF₃, —CN,—C₂R¹, —SO₂CH₃, —C(═O)NR¹R²—NR′C(═O)R¹, —C(═O)R¹, —C(═O)OR¹,—(CH₂)_(q)OR¹, —OC(═O)R¹, —OC(═O)NR¹R², —NR¹(C═Y)—NR¹R², —NR¹(C═Y)—OH,—NR¹(C═Y)—SH, sulfonyl, sulfinyl, phosphoryl, and azo.

wherein any double bond can be in the cis or trans (or E or Z)configuration, and wherein the dashed line is an optional double bond.

Representative compounds falling with the scope of Formula A include:

The following specific compounds are also intended to be encompassed:

where X, T, and Z are as defined elsewhere, and Ar includes theheteroaryl groups in both Ar₃ and Ar₄ as defined elsewhere.

Specific compounds within the scope of Formula A also include:

Additional compounds within the scope of Formula A include thefollowing:

Formulas C-E are shown below:

wherein, for Formulas C-E,

R³ and R⁴ are, individually, H or a substituent J,

R⁵ is selected from H, —CH₂OCH₃, —C(R¹)═X—R⁶, or is a double bondattached to the ring, to a hydrogen, and to CH-T-R⁶,

R⁶ is an aryl or five or six membered ring heteroaryl, optionallysubstituted with from one to three substituents, J, as defined above,

X is CR¹ or N, and

T is C(R¹)₂, NR¹, O or S,

wherein any double bond can be in the cis or trans (or E or Z)configuration, and wherein the dashed line is an optional double bond.

Formulas F-H are shown below

wherein

R³ is selected from —CH₂OCH₃, —C(R¹)═X—R⁴, —CH₂OR⁴—CH₂—R⁴ or is a doublebond attached to the ring, to a hydrogen, and to -T-R⁴,

R⁴ is an aryl or five or six membered ring heteroaryl, optionallysubstituted with from one to three substituents,

J, is a non-hydrogen substituent selected from the group consisting ofhalo (—F, —Cl, —Br, —I), nitro, amino (NR¹R²), OR¹, SR¹, —R¹, —CF₃, —CN,—C₂R, —SO₂CH₃, —C(═O)NR¹R²—NR′C(═O)R¹, —C(═O)R¹, —C(═O)OR¹,—(CH₂)_(q)OR¹, —OC(═O)R¹, —OC(═O)NR¹R², —NR¹(C═Y)—NR¹R², —NR¹(C═Y)—OH,—NR¹(C═Y)—SH, sulfonyl, sulfinyl, phosphoryl, and azo.

X is CR¹ or N (where R¹ is defined above), and

T is, independently, C(R¹)₂, NR¹, O or S,

wherein any double bond can be in the cis or trans configuration, andwherein the dashed line is an optional double bond.

Formulas I and J are shown below:

wherein R¹ is as defined above,

R⁷ is selected from the group consisting of H, —C₁₋₆ alkyl, —C₁₋₆substituted alkyl, —C₆₋₁₀ aryl, —C₆₋₁₀ substituted aryl, —C₆₋₁₀heteroaryl, —C₆₋₁₀ substituted heteroaryl, —C(O)-alkyl,—C(O)-substituted alkyl, —C(O)-aryl, —C(O)-substituted aryl,—C(O)-arylalkyl, C(O)-substituted arylalkyl, —C(O)-alkylaryl,—C(O)-substituted alkylaryl-CN, N₃, NO₂, —OH, —NH₂, —SH, —OR¹, —NHR¹,—N(R)₂, —SR¹, —OC(O)R¹, NHC(O)R¹, —SC(O)R¹, —OC(O)OR¹, —NHC(O)R¹,—CH₂OH, —CH₂CN, —CH₂N₃, —CO₂R¹, —CON(R¹)₂, —C(O)-alkyl,—C(O)-substituted alkyl, —C(O)-aryl, —C(O)-substituted aryl,—C(O)-heteroaryl, and —C(O)— substituted heteroaryl,

R⁸⁻⁹ are, individually, H, OH, —NH₂, —SH, —OR¹, —NHR¹, —N(R¹)₂, —SR¹,—OC(O)R¹, NHC(O)R¹, —C₁₋₆ alkyl, —C₁₋₆ substituted alkyl, —C₆₋₁₀ aryl,—C₆₋₁₀ substituted aryl, —C₆₋₁₀ heteroaryl, —C₆₋₁₀ substitutedheteroaryl,

wherein R¹ is as defined above,

R¹ is as defined above,

R⁷ is selected from the group consisting of H, —C₁₋₆ alkyl, —C₁₋₆substituted alkyl, —C₆₋₁₀ aryl, —C₆₋₁₀ substituted aryl, —C₆₋₁₀heteroaryl, —C₆₋₁₀ substituted heteroaryl, —C(O)-alkyl,—C(O)-substituted alkyl, —C(O)-aryl, —C(O)-substituted aryl,—C(O)-arylalkyl, C(O)-substituted arylalkyl, —C(O)-alkylaryl,—C(O)-substituted alkylaryl-CN, N₃, NO₂, —OH, —NH₂, —SH, —OR¹, —NHR¹,—N(R)₂, —SR¹, —OC(O)R¹, NHC(O)R¹, —SC(O)R¹, —OC(O)OR¹, —NHC(O)R¹,—CH₂OH, —CH₂CN, —CH₂N₃, —CO₂R¹, —CON(R)₂, —C(O)-alkyl, —C(O)-substitutedalkyl, —C(O)-aryl, —C(O)-substituted aryl, —C(O)-heteroaryl, and —C(O)—substituted heteroaryl,

R¹⁰ is —CH₂—, —S—, —O—, —NHR¹—, —N(R¹)₂—, —C(O)—, —C(S)—, —C(NR¹)₂—,

N is 0-4, and

Z is a substituent as defined elsewhere herein.

Specific compounds within formulas I and J are provided below:

Further specific compounds include:

Still further additional compounds include the following:

Enantiomers

The compounds described herein may have asymmetric centers and occur asracemates, racemic mixtures, individual diastereomers or enantiomers,with all isomeric forms being included in the present invention.Compounds of the present invention having a chiral center can exist inand be isolated in optically active and racemic forms. Some compoundscan exhibit polymorphism. The present invention encompasses racemic,optically-active, polymorphic, or stereoisomeric forms, or mixturesthereof, of a compound of the invention, which possess the usefulproperties described herein.

In certain embodiments, the compounds are present as enantiomers. In oneembodiment, the compound is provided as an enantiomer or mixture ofenantiomers. In a particular embodiment, the compound is present as aracemic mixture. The enantiomer can be named by the configuration at thechiral center, such as R or S. In certain embodiments, the compound ispresent as a racemic mixture of R- and S-enantiomers. In certainembodiments, the compound is present as a mixture of two enantiomers. Inone embodiment, the mixture has an enantiomeric excess in R. In oneembodiment, the mixture has an enantiomeric excess in S. In certainother embodiments, the compound is in an enantiomeric excess of the R-or S-enantiomer. The enantiomeric excess can be 51% or more, such as 51%or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% ormore, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more,or 99% or more in the single enantiomer. The enantiomeric excess can be51% or more, such as 51% or more, 55% or more, 60% or more, 65% or more,70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 98% or more, or 99% or more in the R enantiomer. The enantiomericexcess can be 51% or more, such as 51% or more, 55% or more, 60% ormore, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more,90% or more, 95% or more, 98% or more, or 99% or more in the Senantiomer.

In other embodiments, the compound is substantially in the form of asingle enantiomer, such as the R or S enantiomer. The phrase“substantially in the form of a single enantiomer” is intended to meanat least 70% or more in the form of a single enantiomer, for example 70%or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 98% or more, or 99% or more in either the R or S enantiomer.

The enantiomer can be named by the direction in which it rotates theplane of polarized light. If it rotates the light clockwise as seen bythe viewer towards whom the light is traveling, the isomer can belabeled (+) and if it rotates the light counterclockwise, the isomer canbe labeled (−). In certain embodiments, the compound is present as aracemic mixture of (+) and (−) isomers. In certain embodiments, thecompound is present as a mixture of two isomers. In one embodiment, themixture has an excess in (+). In one embodiment, the mixture has anexcess in (−). In certain other embodiments, the compound is in anexcess of the (+) or (−) isomer. The isomeric excess can be 51% or more,such as 51% or more, 55% or more, 60% or more, 65% or more, 70% or more,75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% ormore, or 99% or more in the (+) isomer. The enantiomeric excess can be51% or more, such as 51% or more, 55% or more, 60% or more, 65% or more,70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 98% or more, or 99% or more in the (−) isomer.

In other embodiments, the compound is substantially in the form of asingle optical isomer, such as the (+) or (−) isomer. The phrase“substantially in the form of a single optical isomer” is intended tomean at least 70% or more in the form of a single isomer, for example70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 98% or more, or 99% or more of either the (+) or (−) isomer.

The optically active forms can be prepared by, for example, resolutionof the racemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase or byenzymatic resolution.

Optically active forms of the compounds can be prepared using any methodknown in the art, including but not limited to by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase.

Examples of methods to obtain optically active materials include atleast the following.

-   -   i) physical separation of crystals: a technique whereby        macroscopic crystals of the individual enantiomers are manually        separated. This technique can be used if crystals of the        separate enantiomers exist, i.e., the material is a        conglomerate, and the crystals are visually distinct;    -   ii) simultaneous crystallization: a technique whereby the        individual enantiomers are separately crystallized from a        solution of the racemate, possible only if the latter is a        conglomerate in the solid state;    -   iii) enzymatic resolutions: a technique whereby partial or        complete separation of a racemate by virtue of differing rates        of reaction for the enantiomers with an enzyme;    -   iv) enzymatic asymmetric synthesis: a synthetic technique        whereby at least one step of the synthesis uses an enzymatic        reaction to obtain an enantiomerically pure or enriched        synthetic precursor of the desired enantiomer;    -   v) chemical asymmetric synthesis: a synthetic technique whereby        the desired enantiomer is synthesized from an achiral precursor        under conditions that produce asymmetry (i.e., chirality) in the        product, which can be achieved using chiral catalysts or chiral        auxiliaries;    -   vi) diastereomer separations: a technique whereby a racemic        compound is reacted with an enantiomerically pure reagent (the        chiral auxiliary) that converts the individual enantiomers to        diastereomers. The resulting diastereomers are then separated by        chromatography or crystallization by virtue of their now more        distinct structural differences and the chiral auxiliary later        removed to obtain the desired enantiomer;    -   vii) first- and second-order asymmetric transformations: a        technique whereby diastereomers from the racemate equilibrate to        yield a preponderance in solution of the diastereomer from the        desired enantiomer or where preferential crystallization of the        diastereomer from the desired enantiomer perturbs the        equilibrium such that eventually in principle all the material        is converted to the crystalline diastereomer from the desired        enantiomer. The desired enantiomer is then released from the        diastereomer;    -   viii) kinetic resolutions: this technique refers to the        achievement of partial or complete resolution of a racemate (or        of a further resolution of a partially resolved compound) by        virtue of unequal reaction rates of the enantiomers with a        chiral, non-racemic reagent or catalyst under kinetic        conditions;    -   ix) enantiospecific synthesis from non-racemic precursors: a        synthetic technique whereby the desired enantiomer is obtained        from non-chiral starting materials and where the stereochemical        integrity is not or is only minimally compromised over the        course of the synthesis;    -   x) chiral liquid chromatography: a technique whereby the        enantiomers of a racemate are separated in a liquid mobile phase        by virtue of their differing interactions with a stationary        phase (including but not limited to via chiral HPLC). The        stationary phase can be made of chiral material or the mobile        phase can contain an additional chiral material to provoke the        differing interactions;    -   xi) chiral gas chromatography: a technique whereby the racemate        is volatilized and enantiomers are separated by virtue of their        differing interactions in the gaseous mobile phase with a column        containing a fixed non-racemic chiral adsorbent phase;    -   xii) extraction with chiral solvents: a technique whereby the        enantiomers are separated by virtue of preferential dissolution        of one enantiomer into a particular chiral solvent;    -   xiii) transport across chiral membranes: a technique whereby a        racemate is placed in contact with a thin membrane barrier. The        barrier typically separates two miscible fluids, one containing        the racemate, and a driving force such as concentration or        pressure differential causes preferential transport across the        membrane barrier. Separation occurs as a result of the        non-racemic chiral nature of the membrane that allows only one        enantiomer of the racemate to pass through.

Chiral chromatography, including but not limited to simulated moving bedchromatography, is used in one embodiment. A wide variety of chiralstationary phases are commercially available.

II. Methods of Preparing the Compounds

General synthetic methods for preparing the compounds described hereinare provided below. These synthetic methods are not intended to belimiting. Those of skill in the art are well aware of means forproviding various functional groups, derivatives, and protecting groupson aromatic rings and other moieties, and can readily adapt thesegeneral methods to synthesize the compounds described herein.

Synthesis of Compounds of Formula A

The compounds of Formula A (also referred to herein as the 1180 class)are members of the class of substituted tetrahydroisoquinolines.Synthesis of these compounds can proceed via two routes. Scheme 5outlines the synthetic route proceeding through a Bischler-Napieralskicyclization. An appropriately derivatized phenethylamine (1) (orvariation thereof with a 5- or 6-membered ring heteroaryl ring in placeof the aryl ring) is allowed to react with carboxylic acid 2, oractivated derivative thereof, such as an acid halide, to form thecorresponding amide (3).

The amide formed is then subjected to a dehydrating agent, such as POCl₃and/or P₂O₅ to form a dihydroisoquinoline (4) through aBischler-Napieralski reaction. The newly-formed dihydroisoquinoline canthen be reduced with an appropriate hydride source, such as NaBH₄, tothe subsequent tetrahydroisoquinoline (5). An appropriate chiral hydridesource could provide access to optically-enriched or optically-pureproduct. Alternatively, enantiomers can be obtained using a variety ofresolution techniques that are well known to those skilled in the art.The tetrahydroisoquinoline is then allowed to react with anappropriately substituted acyl chloride, activated carboxylic acid, oralkyl halide.

Although the aryl rings in Scheme 1 are shown as phenyl, the chemistryworks with other aryl and heteroaryl rings, as described herein, so longas the J substituents either do not react with the functional groupsinvolved in the coupling chemistry, or the J substituents are suitablyprotected so that they do not so interfere.

In Scheme 1, R₁-R₆ are independently H, OMe, OH, SH, SMe, Cl, F, I, Br,C₁₋₆ alkyl, C₆₋₁₀ aryl, alkylaryl, and arylalkyl, and substitutedversions of the alkyl, aryl, aralkyl, and alkylaryl moieties, as definedherein, X═NH, NR where R is an alkyl or aryl substituent, O, S, CH2, orCHR where R is C₁₋₆ alkyl, C₆₋₁₀ aryl, alkylaryl, and arylalkyl, andsubstituted versions of the alkyl, aryl, aralkyl, and alkylarylmoieties, as defined herein, Y═S, NH, NR (where R is same as above),CH2, CR2, CHR, or O.

In Scheme 2, an appropriately functionalized phenethylamine (1) isallowed to react with an aldehyde (7) to form the imine 8. The imine isthen cyclized via Pictet-Spengler reaction conditions to form thetetrahydroisoquinoline 5. The tetrahydroisoquinoline is then allowed toreact with an appropriately substituted acid chloride, activatedcarboxylic acid, or alkyl halide.

The various substituents in Scheme 2 are defined in the same manner asin Scheme 1.

Representative syntheses of compounds of Formulas I and J are shownbelow:

Cyclic ketones 1 are reacted with ethyl cyanoacetate 2 and sulfur with acatalytic amount of morpholine in ethanol to give amino thiophenes 3 ingenerally moderate to good yields. Compounds 3 will be reacted with acylchlorides to give amides 4 in moderate to excellent yields. Esterhydrolysis

Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxyate

-   Andersen, H. S.; Olsen, O. H.; Iversen, L. F.; Sørensen, A. L. P.;    Mortensen, S. B.; Christensen, M. S.; Branner, S.; Hansen, T. K.;    Lau, J. F.; Jeppesen, L.; Moran, E. J.; Su, J.; Bakir, F.; Judge,    L.; Shahbaz, M.; Collins, T.; Vo, T.; Newman, M. J.; Ripka, W. C.;    Møller, N. P. H. J. Med. Chem. 2002, 45, 4443-4459.

To a solution of cyclohexanone (5.0 g, 51 mmol), ethylcyanoacetate (5.96mL, 56 mmol, 1.1 equiv) and sulfur (1.80 g, 56 mmol, 1.1 equiv) inabsolute ethanol (150 mL), was added morpholine (6.7 mL, 76 mmol, 1.5equiv). The yellow mixture was heated to 50° C. for 16 h. The brownishsolution was evaporated and the resulting solid was dissolved in ethylacetate (100 mL), washed with water (2×50 mL) and brine (2×50 mL) anddried over magnesium sulfate. The solvent was evaporated and the crudeproduct was subjected to purification by chromatography on silica gelcolumn, using hexanes/ethyl acetate 10:1 v/v as the eluent. The expectedcompound was isolated as an off-white powder (10.40 g, 91%).

Ethyl2-(3-(trifluoromethyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

-   Pittallá, V; Modica, M.; Romeo, G.; Materia, L.; Salerno, L.;    Siracusa, M.; Cagnotto, A.; Merghetti, I.; Russo, F. Il Farmaco    2005, 60, 711-720

A solution of 3-trifluorobenzoyl chloride (0.300 g, 1.33 mmol), ethyl2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (200 mL, 1.33mmol, 1 equiv) and triethylamine (260 mL, 1.86 mmol, 1.4 equiv) indichloromethane (5.4 mL) was stirred for 2 h at room temperature. Thereaction mixture was then diluted with dichloromethane. The organiclayer was washed with an aqueous 1N solution of sodium hydroxide (3×10mL) and brine (10 mL), dried over magnesium sulfate, filtered andevaporated under reduced pressure. The crude product was purified bysilica gel chromatography using hexanes/ethyl acetate 10:1 v/v as theeluent. The expected compound was obtained as a yellow solid (0.455 g,86%).

2-(3-(Trifluoromethyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylicacid

-   Pittallá, V; Modica, M.; Romeo, G.; Materia, L.; Salerno, L.;    Siracusa, M.; Cagnotto, A.; Merghetti, L; Russo, F. Il Farmaco 2005,    60, 711-720

A solution of the previous ethyl ester (0.300 g, 0.75 mmol) was added toa 2N aqueous solution of sodium hydroxide (1.6 mL) in ethanol (8 mL).The mixture was heated at 90° C. for 2 h. After cooling, the mixture wasacidified with concentrated aqueous hydrochloric acid and theprecipitate was filtered and washed with cold water to afford theexpected product as a white solid (0.170 g, 61%).

The intention of this application is to encompass all existingstereoisomers of all compounds present. Methods to prepare or resolve,isolate and evaluate the individual isomers are well known by thoseskilled in the art. These approaches include the use of chiralauxiliaries, chiral catalysts, kinetic resolution, chiralhigh-performance liquid chromatography (HPLC), classical resolution, andthe like (Gremmen et al 2001; Kanemitsu et al 2006; Paal et al 2008;Piwowarczyk et al 2008; Schuster et al 2007).

III. Pharmaceutical Compositions Including the Compounds

Mammals, and specifically humans, suffering from schizophrenia,Parkinson's disease, depression, neuropathic pain, stroke, traumaticbrain injury, epilepsy, and other neurologic events or neurodegenerationinvolving NMDA receptor activation, or any of the above-describedconditions, can be treated by either targeted or systemicadministration, via oral, inhalation, topical, trans- or sub-mucosal,subcutaneous, parenteral, intramuscular, intravenous or transdermaladministration of a composition comprising an effective amount of thecompounds described herein or a pharmaceutically acceptable salt, esteror prodrug thereof, optionally in a pharmaceutically acceptable carrier.The compounds or composition is typically administered by oraladministration. Alternatively, compounds can be administered byinhalation. In another embodiment, the compound is administeredtransdermally (for example via a slow release patch), or topically. Inyet another embodiment, the compound is administered subcutaneously,intravenously, intraperitoneally, intramuscularly, parenterally, orsubmucosally. In any of these embodiments, the compound is administeredin an effective dosage range to treat the target condition.

In one embodiment, compounds of the present invention are administeredorally. Oral compositions will generally include an inert diluent or anedible carrier. They may be enclosed in gelatin capsules or compressedinto tablets. For the purpose of oral therapeutic administration, theactive compound can be incorporated with excipients and used in the formof tablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

When the compound is administered orally in the form of a dosage unitsuch as a tablets, pills, capsules, troches and the like, these cancontain any of the following ingredients, or compounds of a similarnature: a binder (such as microcrystalline cellulose, gum tragacanth orgelatin); an excipient (such as starch or lactose), a disintegratingagent (such as alginic acid, Primogel, or corn starch); a lubricant(such as magnesium stearate or Sterotes); a glidant (such as colloidalsilicon dioxide); a sweetening agent (such as sucrose or saccharin);and/or a flavoring agent (such as peppermint, methyl salicylate, ororange flavoring). When the dosage unit form is a capsule, it cancontain, in addition to material of the above type, a liquid carrier(such as a fatty oil). In addition, dosage unit forms can containvarious other materials which modify the physical form of the dosageunit, for example, coatings of sugar, shellac, or other enteric agents.The compound or its salts can also be administered orally as a componentof an elixir, suspension, syrup, wafer, chewing gum or the like. A syrupmay contain, in addition to the active compounds, a sweetening agent(such as sucrose, saccharine, etc.) and preservatives, dyes andcolorings and flavors.

The compounds of the invention may be also administered in specific,measured amounts in the form of an aqueous suspension by use of a pumpspray bottle. The aqueous suspension compositions of the presentinvention may be prepared by admixing the compounds with water and otherpharmaceutically acceptable excipients. The aqueous suspensioncompositions according to the present invention may contain, inter alia,water, auxiliaries and/or one or more of the excipients, such as:suspending agents, e.g., microcrystalline cellulose, sodiumcarboxymethylcellulose, hydroxpropyl-methyl cellulose; humectants, e.g.glycerin and propylene glycol; acids, bases or buffer substances foradjusting the pH, e.g., citric acid, sodium citrate, phosphoric acid,sodium phosphate as well as mixtures of citrate and phosphate buffers;surfactants, e.g. Polysorbate 80; and antimicrobial preservatives, e.g.,benzalkonium chloride, phenylethyl alcohol and potassium sorbate. In aseparate embodiment, the compounds of the invention are in the form ofan inhaled dosage. In this embodiment, the compounds may be in the formof an aerosol suspension, a dry powder or liquid particle form. Thecompounds may be prepared for delivery as a nasal spray or in aninhaler, such as a metered dose inhaler. Pressurized metered-doseinhalers (“MDI”) generally deliver aerosolized particles suspended inchlorofluorocarbon propellants such as CFC-11, CFC-12, or thenon-chlorofluorocarbons or alternate propellants such as thefluorocarbons, HFC-134A or HFC-227 with or without surfactants andsuitable bridging agents. Dry-powder inhalers can also be used, eitherbreath activated or delivered by air or gas pressure such as thedry-powder inhaler disclosed in the Schering Corporation InternationalPatent Application No. PCT/US92/05225, published 7 Jan. 1993 as well asthe Turbuhaler™ (available from Astra Pharmaceutical Products, Inc.) orthe Rotahaler™ (available from Allen & Hanburys) which may be used todeliver the aerosolized particles as a finely milled powder in largeaggregates either alone or in combination with some pharmaceuticallyacceptable carrier e.g. lactose; and nebulizers. Solutions orsuspensions used for parenteral, intradermal, subcutaneous, or topicalapplication can include at least some of the following components: asterile diluent (such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents); antibacterial agents (such as benzyl alcohol ormethyl parabens); antioxidants (such as ascorbic acid or sodiumbisulfite); chelating agents (such as ethylenediaminetetraacetic acid);buffers (such as acetates, citrates or phosphates); and/or agents forthe adjustment of tonicity (such as sodium chloride or dextrose). The pHof the solution or suspension can be adjusted with acids or bases, suchas hydrochloric acid or sodium hydroxide.

A parenteral preparation can be enclosed in ampoules, disposablesyringes or multiple dose vials made of glass or plastic.

Suitable vehicles or carriers for topical application can be prepared byconventional techniques, such as lotions, suspensions, ointments,creams, gels, tinctures, sprays, powders, pastes, slow-releasetransdermal patches, suppositories for application to rectal, vaginal,nasal or oral mucosa. In addition to the other materials listed abovefor systemic administration, thickening agents, emollients, andstabilizers can be used to prepare topical compositions. Examples ofthickening agents include petrolatum, beeswax, xanthan gum, orpolyethylene, humectants such as sorbitol, emollients such as mineraloil, lanolin and its derivatives, or squalene.

If administered intravenously, carriers can be physiological saline,bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In one embodiment, the active compoundsare prepared with carriers that will protect the compound against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)are also preferred as pharmaceutically acceptable carriers. These may beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811 (which is incorporatedherein by reference in its entirety). For example, liposome formulationsmay be prepared by dissolving appropriate lipid(s) (such as stearoylphosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoylphosphatidyl choline, and cholesterol) in an inorganic solvent that isthen evaporated, leaving behind a thin film of dried lipid on thesurface of the container. An aqueous solution of the compound is thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension.

Dosing

The compound is administered for a sufficient time period to alleviatethe undesired symptoms and the clinical signs associated with thecondition being treated. In one embodiment, the compounds areadministered less than three times daily. In one embodiment, thecompounds are administered in one or two doses daily. In one embodiment,the compounds are administered once daily. In some embodiments, thecompounds are administered in a single oral dosage once a day. Theactive compound is included in the pharmaceutically acceptable carrieror diluent in an amount sufficient to deliver to a patient a therapeuticamount of compound in vivo in the absence of serious toxic effects. Aneffective dose can be readily determined by the use of conventionaltechniques and by observing results obtained under analogouscircumstances. In determining the effective dose, a number of factorsare considered including, but not limited to: the species of patient;its size, age, and general health; the specific disease involved; thedegree of involvement or the severity of the disease; the response ofthe individual patient; the particular compound administered; the modeof administration; the bioavailability characteristics of thepreparation administered; the dose regimen selected; and the use ofconcomitant medication.

Typical systemic dosages for the herein described conditions are thoseranging from 0.01 mg/kg to 1500 mg/kg of body weight per day as a singledaily dose or divided daily doses. Preferred dosages for the describedconditions range from 0.5-1500 mg per day. A more particularly preferreddosage for the desired conditions ranges from 5-750 mg per day. Typicaldosages can also range from 0.01 to 1500, 0.02 to 1000, 0.2 to 500, 0.02to 200, 0.05 to 100, 0.05 to 50, 0.075 to 50, 0.1 to 50, 0.5 to 50, 1 to50, 2 to 50, 5 to 50, 10 to 50, 25 to 50, 25 to 75, 25 to 100, 100 to150, or 150 or more mg/kg/day, as a single daily dose or divided dailydoses. In one embodiment, the daily dose is between 10 and 500 mg/day.In another embodiment, the dose is between about 10 and 400 mg/day, orbetween about 10 and 300 mg/day, or between about 20 and 300 mg/day, orbetween about 30 and 300 mg/day, or between about 40 and 300 mg/day, orbetween about 50 and 300 mg/day, or between about 60 and 300 mg/day, orbetween about 70 and 300 mg/day, or between about 80 and 300 mg/day, orbetween about 90 and 300 mg/day, or between about 100 and 300 mg/day, orabout 200 mg/day. In one embodiment, the compounds are given in doses ofbetween about 1 to about 5, about 5 to about 10, about 10 to about 25 orabout 25 to about 50 mg/kg. Typical dosages for topical application arethose ranging from 0.001 to 100% by weight of the active compound.

The concentration of active compound in the drug composition will dependon absorption, inactivation, and excretion rates of the drug as well asother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition. Theactive ingredient may be administered at once, or may be divided into anumber of smaller doses to be administered at varying intervals of time.

Combination Treatment

The compound can also be mixed with other active materials which do notimpair the desired action, or with materials that supplement the desiredaction. The active compounds can be administered in conjunction, i.e.combination or alternation, with other medications used in the treatmentor prevention schizophrenia, Parkinson's disease, depression,neuropathic pain, stroke, traumatic brain injury, epilepsy, as well asother neurologic events, neurological disorders, or neurodegenerativeconditions. In another embodiment, the compounds can be administered inconjunction (combination or alternation) with other medications used intreatment or prophylaxis of inflammatory conditions. In certainembodiments, the combination can be synergistic although in otherembodiments the combination is not synergistic.

IV. Methods of Treatment Using the Compounds

In one embodiment, the compounds are used in a method of treatment orprophylaxis of schizophrenia, bipolar disorder, depression, anxiety,neuropsychiatric or mood disorders, obsessive-compulsive disorder,neurocognitive disorders, pre-senile dementia, motor dysfunction ormotor disorders, tardive dyskinesia, neuropathic pain, inflammatorypain, Parkinson's disease, Alzheimer's disease, amyolateral sclerosis(ALS), Huntington's chorea, epilepsy, traumatic brain injury, ischemicand hemorrhagic stroke, subarachnoid hemorrhage, cerebral vasospasm,ischemia, hypoxia, or neurodegeneration involving NMDA receptoractivation comprising administering to a host in need thereof aneffective amount of a compound of any of Claims 1-9, optionally in apharmaceutically acceptable carrier. The compounds can be administered,alone or in a pharmaceutically-acceptable carrier, to treat, prevent, orreduce the symptoms of the various disorders.

The compounds described herein can also generally be used to treatneurologic events and neurodegeneration, whether or not such neurologicevent or neurodegeneration is associated with NMDA receptor activation.

The compounds described herein can also generally be used to treatpatients who are under the actions of an NMDA receptor antagonists suchas PCP or ketamine.

In some embodiments, the compounds are used to treat or prevent strokeor stroke damage, and can be administered under emergency care for astroke, for maintenance treatment of stroke, and/or for rehabilitationof stroke.

In other embodiments, the compounds are used to provide cognitiveenhancement, in normal or cognitively deficient individuals.

In another embodiment, the compounds are used to improve rehabilitativetraining after stroke, head injury, ischemia, hypoxia, or any acutebrain injury.

In one embodiment, methods are provided to treat patients with ischemicinjury or hypoxia, or prevent or treat the neuronal toxicity associatedwith ischemic injury or hypoxia, by administering a compound orcomposition described herein. In one aspect of this embodiment, theischemic injury is vasospasm after subarachnoid hemorrhage.

A subarachnoid hemorrhage refers to an abnormal condition in which bloodcollects beneath the arachnoid mater, a membrane that covers the brain.This area, called the subarachnoid space, normally containscerebrospinal fluid. The accumulation of blood in the subarachnoid spaceand the vasospasm of the vessels which results from it can lead tostroke, seizures, and other complications. The methods and compoundsdescribed herein can be used to treat patients experiencing asubarachnoid hemorrhage. In one embodiment, the methods and compoundsdescribed herein can be used to limit the toxic effects of thesubarachnoid hemorrhage, including, for example, stroke and/or ischemiathat can result from the subarachnoid hemorrhage. In a particularembodiment, the methods and compounds described herein can be used totreat patients with traumatic subarachnoid hemorrhage. On oneembodiment, the traumatic subarachnoid hemorrhage can be due to a headinjury. In another embodiment, the patients can have a spontaneoussubarachnoid hemorrhage.

In other embodiments, the ischemic injury is selected from, but notlimited to, one of the following: traumatic brain injury, cognitivedeficit after traumatic brain injury or cerebral ischemia, cognitivedeficit after cerebral hypoxia, cognitive deficit after bypass surgery,cognitive deficit after carotid angioplasty; and cognitive deficit afterneonatal ischemia following hypothermic circulatory arrest.

In another embodiment, methods are provided to treat patients with braintumors, such as gliomas, by administering a compound selected accordingto the methods or processes described herein.

Further, the methods described herein can be used prophylactically toprevent or protect against such diseases or neurological conditions,such as those described herein. In one embodiment, patients with apredisposition for an ischemic event, such as a genetic predisposition,can be treated prophylactically with the methods and compounds describedherein. In another embodiment, patients that exhibit vasospasms can betreated prophylactically with the methods and compounds describedherein. In a further embodiment, patients that have undergone cardiacbypass surgery can be treated prophylactically with the methods andcompounds described herein.

In one embodiment, methods are provided to treat patients with ischemicinjury or hypoxia, or prevent or treat the neuronal toxicity associatedwith ischemic injury or hypoxia, by administering a compound selectedaccording to the methods or processes described herein.

In another embodiment, methods are provided to treat patients withinflammatory pain or neuropathic pain or related disorders byadministering a compound selected according to the methods or processesdescribed herein. In certain embodiments, the neuropathic pain orrelated disorder can be selected from the group including, but notlimited to: peripheral diabetic neuropathy, postherpetic neuralgia,complex regional pain syndromes, peripheral neuropathies,chemotherapy-induced neuropathic pain, cancer neuropathic pain,neuropathic low back pain, HIV neuropathic pain, trigeminal neuralgia,and/or central post-stroke pain.

Neuropathic pain can be associated with signals generated ectopicallyand often in the absence of ongoing noxious events by pathologicprocesses in the peripheral or central nervous system. This dysfunctioncan be associated with common symptoms such as allodynia, hyperalgesia,intermittent abnormal sensations, and spontaneous, burning, shooting,stabbing, paroxysmal or electrical-sensations, paresthesias, hyperpathiaand/or dysesthesias, which can also be treated by the compounds andmethods described herein. Further, the compounds and methods describedherein can be used to treat neuropathic pain resulting from peripheralor central nervous system pathologic events, including, but not limitedto trauma, ischemia; infections or from ongoing metabolic or toxicdiseases, infections or endocrinologic disorders, including, but notlimited to, diabetes mellitus, diabetic neurophathy, amyloidosis,amyloid polyneuropathy (primary and familial), neuropathies withmonoclonal proteins, vasculitic neuropathy, HIV infection, herpeszoster—shingles and/or postherpetic neuralgia; neuropathy associatedwith Guillain-Barre syndrome; neuropathy associated with Fabry'sdisease; entrapment due to anatomic abnormalities; trigeminal and otherCNS neuralgias; malignancies; inflammatory conditions or autoimmunedisorders, including, but not limited to, demyelinating inflammatorydisorders, rheumatoid arthritis, systemic lupus erythematosus, Sjogren'ssyndrome; and cryptogenic causes, including, but not limited toidiopathic distal small-fiber neuropathy. Other causes of neuropathicpain that can be treated according to the methods and compositionsdescribed herein include, but are not limited to, exposure to toxins ordrugs (such as aresnic, thallium, alcohol, vincristine, cisplatinum anddideoxynucleosides), dietary or absorption abnormalities,immuno-globulinemias, hereditary abnormalities and amputations(including mastectomy). Neuropathic pain can also result fromcompression of nerve fibers, such as radiculopathies and carpal tunnelsyndrome.

The compounds can also be used to treat the following diseases orneurological conditions, including, but not limited to: chronic nerveinjury, chronic pain syndromes, such as, but not limited to ischemiafollowing transient or permanent vessel occlusion, seizures, spreadingdepression, restless leg syndrome, hypocapnia, hypercapnia, diabeticketoacidosis, fetal asphyxia, spinal cord injury, status epilepticus,concussion, migraine, hypocapnia, hyperventilation, lactic acidosis,fetal asphyxia during parturition, and/or retinopathies by administeringa compound selected according to the methods or processes describedherein.

In one embodiment, the use of the compounds of the invention reducessymptoms of neuropathic pain, stroke, traumatic brain injury, epilepsy,and other neurologic events or neurodegeneration resulting from NMDAreceptor activation.

Alzheimer's Disease

Senile dementia of the Alzheimer's type (SDAT) is a debilitatingneurodegenerative disease, mainly afflicting the elderly, characterizedby a progressive intellectual and personality decline, as well as a lossof memory, perception, reasoning, orientation and judgment. One featureof the disease is an observed decline in the function of cholinergicsystems, and specifically, a severe depletion of cholinergic neurons(i.e., neurons that release acetylcholine, which is believed to be aneurotransmitter involved in learning and memory mechanisms). See, forexample, Jones et al., Intern. J. Neurosci. 50:147 (1990); Perry, Br.Med. Bull. 42:63 (1986); and Sitaram et al., Science 201:274 (1978).

A dysfunction of glutamatergic neurotransmission is hypothesized to beinvolved in the etiology of Alzheimer's disease. Targeting theglutamatergic system, specifically NMDA receptors, offers a novelapproach to treatment in view of the limited efficacy of existing drugstargeting the cholinergic system. Cacabelos R, Takeda M, Winblad B(January 1999). “The glutamatergic system and neurodegeneration indementia: preventive strategies in Alzheimer's disease”. Int J Geriatr.Psychiatry 14 (1):3-47. By binding to the NMDA receptor the NMDAreceptor potentiators described herein are able to enhance the prolongedinflux of Ca²⁺ ions which forms the basis of neuronal plasticity, and isthought to be involved in learning and memory. In addition, glutamatereceptors are intimately involved in the molecular substrates ofcognition, learning and memory formation. Glutamate receptor modulatorshave been hypothesized to be capable of influencing cognition and memoryformation. Thus, manipulation of the glutamate system bysubunit-selective NMDA receptor potentiators described here couldprovide beneficial relief to patients suffering from Alzheimer's, otherforms of dementia, as well as other neurological conditions that involveimpaired judgment, memory, or cognition.

Parkinson's Disease

Parkinson's disease (PD) is a debilitating neurodegenerative disease,presently of unknown etiology, characterized by tremors, muscularrigidity. A feature of the disease appears to involve the progressivedegeneration of dopaminergic neurons (i.e., which secrete dopamine). Onesymptom of the disease has been observed to be a concomitant loss ofnicotinic receptors which are associated with such dopaminergic neurons,and which are believed to modulate the process of dopamine secretion.See Rinne et al., Brain Res. 54:167 (1991) and Clark et al., Br. J.Pharm. 85:827 (1985).

N-Methyl-D-aspartate (NMDA) glutamate receptors are a class ofexcitatory amino acid receptors, which have several important functionsin the motor circuits of the basal ganglia, and are viewed as importanttargets for the development of new drugs to prevent or treat Parkinson'sdisease (PD). NMDA receptors are ligand-gated ion channels composed ofmultiple subunits, each of which has distinct cellular and regionalpatterns of expression. They have complex regulatory properties, withboth agonist and co-agonist binding sites and regulation byphosphorylation and protein-protein interactions. They are found in allof the structures of the basal ganglia, although the subunit compositionin the various structures is different. NMDA receptors present in thestriatum are crucial for dopamine-glutamate interactions. The abundance,structure, and function of striatal receptors are altered by thedopamine depletion and further modified by the pharmacologicaltreatments used in PD. Given the expression of NR2C and NR2D subunits onkey neurons such as the dopamine releasing substantia nigra parscompacta neurons, it is possible that the subunit-selective NMDAreceptor potentiators described here could find some use in cognitiveeffects of Parkinson's disease, or in the enhancement of dopaminesignalling.

Tardive Diskinesia and Other Motor Disorders

In one embodiment, the invention relates to a method of treating tardivedyskinesia in humans.

In one aspect, the invention reduces involuntary movements orhyperkinesia characteristic of patients with tardive movement disorders,including tardive dyskinesia, by administering an NMDA receptorpotentiator as defined herein. The NMDA receptor potentiators could alsobe useful for the treatment of other motor disorders ranging fromresting tremor to various dyskinesias. The NR2C subunit is abundantlyexpressed in the cerebellum, a structure that is involved in fine motorcoordination. Thus, the compounds described here that act on the NR2Csubunit could enhance motor function in a beneficial way for a largenumber of patients.

By enhancing cognition and memory, the compounds described here thatalter the NR2C and NR2D subunit activity could be beneficial infacilitating rehabilitation after brain injury of any type. Suchcompounds might enhance motor reprogramming during physical therapy,thereby increasing functionality and speeding recovery.

In all of these embodiments, the methods involve administering to a hostin need thereof an effective amount of a compound of any of the formulasdescribed herein, or a pharmaceutically acceptable salt, ester, orderivative thereof, or a pharmaceutical composition thereof.

Side Effects

In an additional aspect of the methods and processes described herein,the compound does not exhibit substantial toxic an/or psychotic sideeffects. Toxic side effects include, but are not limited to, agitation,hallucination, confusion, stupor, paranoia, delirium,psychotomimetic-like symptoms, rotorod impairment, amphetamine-likestereotyped behaviors, stereotypy, psychosis memory impairment, motorimpairment, anxiolytic-like effects, increased blood pressure, decreasedblood pressure, increased pulse, decreased pulse, hematologicalabnormalities, electrocardiogram (ECG) abnormalities, cardiac toxicity,heart palpitations, motor stimulation, psychomotor performance, moodchanges, short-term memory deficits, long-term memory deficits, arousal,sedation, extrapyramidal side-effects, ventricular tachycardia, andlengthening of cardiac repolarization, ataxia, cognitive deficits and/orschizophrenia-like symptoms.

In one embodiment, the compound is a selective NR1/NR2C NMDA receptorand/or a NR1/NR2D NMDA receptor potentiator. In one particularembodiment, the compounds can bind to the NR2C or NR2D subunits of theNMDA receptor regardless of the other subunits that are present. Inanother particular embodiment, the compounds are selective for the NR2Cor NR2D subunits of the NMDA receptor. In a further additional oralternative embodiment, the compound has a therapeutic index equal to orgreater than at least 2:1, at least 3:1, at least 4:1, at least 5:1, atleast 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, atleast 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1,at least 50:1, at least 75:1, at least 100:1 or at least 1000:1. Thetherapeutic index can be defined as the ratio of the dose required toproduce toxic or lethal effects to dose required to produce therapeuticresponses. It can be the ratio between the median toxic dose (the dosageat which 50% of the group exhibits the adverse effect of the drug) andthe median effective dose (the dosage at which 50% of the populationrespond to the drug in a specific manner). The higher the therapeuticindex, the more safe the drug is considered to be. It simply indicatesthat it would take a higher dose to invoke a toxic response that it doesto cause a beneficial effect.

The side effect profile of compounds can be determined by any methodknown to those skilled in the art. In one embodiment, motor impairmentcan be measured by, for example, measuring locomotor activity and/orrotorod performance. Rotorod experiments involve measuring the durationthat an animal can remain on an accelerating rod. In another embodiment,memory impairment can be assessed, for example, by using a passiveavoidance paradigm; Sternberg memory scanning and paired words forshort-term memory, or delayed free recall of pictures for long-termmemory. In a further embodiment, anxiolytic-like effects can bemeasured, for example, in the elevated plus maze task. In otherembodiments, cardiac function can be monitored, blood pressure and/orbody temperature measured and/or electrocardiograms conducted to testfor side effects. In other embodiments, psychomotor functions andarousal can be measured, for example by analyzing critical flickerfusion threshold, choice reaction time, and/or body sway. In otherembodiments, mood can be assessed using, for example, self-ratings. Infurther embodiments, schizophrenic symptoms can be evaluated, forexample, using the PANSS, BPRS, and CGI, side-effects were assessed bythe HAS and the S/A scale.

In one embodiment, the compound does not exhibit substantial toxic sideeffects, such as, for example, motor impairment or cognitive impairment.In a particular embodiment, the compound has a therapeutic index equalto or greater than at least 2. In another embodiment, the compound is atleast 10 times more selective for binding to an NMDA receptor than anyother glutamate receptor. In certain embodiments, the compound interactswith hERG channels at an EC₅₀ at least 10 times the EC₅₀ forpotentiation of an NMDA receptor.

Use of NMDA Receptor Potentiators to Inhibit Drug Tolerance andDependence and Assist with Withdrawal, Including Smoking Cessation andOpiate Withdrawal

Potentiators of the NMDA receptor, i.e., compounds that increase thecurrent flow through the channel, can modify cognitive function andpotentially enhance certain forms of learning. By using the compoundsdescribed herein, one can treat tolerance and dependence induced byopiate analgesics, and assist with smoking cessation, without producingunwanted side effects such as schizophrenia-like symptoms, loss ofnormal NMDA receptor-mediated synaptic plasticity (which can possiblyaffect learning and memory), amnesia, confusional states, and musclerelaxation caused by the non-selective NMDA antagonists of the priorart. Thus, the compounds can be used along with opiates to managechronic pain in severely ill patients and alleviate the pain ofwithdrawal both in legitimate and illegitimate drug users.

Use of NMDA Receptor Potentiators with Specificity for the NMDA 2DSubtype in Treating Bone Disorders

Bone formation, or osteogenesis, refers to the creation of new bonemass. This includes the process whereby new bone structure grows or thedensity of existing bone is increased. Osteoblasts form bone byproducing extracellular organic matrix, or osteoid and then mineralizingthe matrix to form bone. The main mineral component of bone iscrystalline hydroxyapetite, which comprises much of the mass of normaladult bone.

In an embodiment of the invention the mammal is a human in need ofenhanced bone formation. In one aspect, the human in need has a bonedeficit, which means that they will have less bone than desirable orthat the bone will be less dense or strong than desired. A bone deficitmay be localized, such as that caused by a bone fracture or systemic,such as that caused by osteoporosis. Bone deficits may result from abone remodeling disorder whereby the balance between bone formation andbone resorption is shifted, resulting in a bone deficit. Examples ofsuch bone remodeling disorders include osteoporosis, Paget's disease,osteoarthritis, rheumatoid arthritis, achondroplasia, osteochodrytis,hyperparathyroidism, osteogenesis imperfecta, congenitalhypophosphatasia, fribromatous lesions, fibrous displasia, multiplemyeloma, abnormal bone turnover, osteolytic bone disease and periodontaldisease. Bone remodelling disorders includes metabolic bone diseaseswhich are characterised by disturbances in the organic matrix, bonemineralization, bone remodelling, endocrine, nutritional and otherfactors which regulate skeletal and mineral homeostasis. Such disordersmay be hereditary or acquired and generally are systemic affecting theentire skeletal system.

In one aspect, the mammal may have a bone remodeling disorder. Boneremodeling as used herein refers to the process whereby old bone isbeing removed and new bone is being formed by a continuous turnover ofbone matrix and mineral that involves bone resorption by osteoclasts andbone formation by osteoblasts.

Osteoporosis is a common bone remodelling disorder characterised by adecrease in bone density of normally mineralised bone, resulting inthinning and increased porosity of bone cortices and trabeculae. Theskeletal fragility caused by osteoporosis predisposes sufferers to bonepain and an increased incidence of fractures. Progressive bone loss inthis condition may result in a loss of up to 50% of the initial skeletalmass.

Primary osteoporosis includes idiopathic osteoporosis which occurs inchildren or young adults with normal gonadal function, Type Iosteoporosis, also described as post-menauposal osteoporosis, and TypeII osteoporosis, senile osteoporosis, occurs mainly in those personsolder than 70 years of age. Causes of secondary osteoporosis may beendocrine (e.g. glucocorticoid excess, hyperparathyroidism,hypoganodism), drug induced (e.g. corticosteroid, heparin, tobaco) andmiscellanous (e.g. chronic renal failure, hepatic disease andmalabsorbtion syndrome osteoporosis). The phrase “at risk of developinga bone deficit”; as used herein, is intended to embrace mammals andhumans having a higher than average predisposition towards developing abone deficit. As an example, those susceptible towards osteoporosisinclude post-menopausal women, elderly males (e.g. those over the age of65) and those being treated with drugs known to cause osteoporosis as aside-effect (e.g. steroid-induced osteoporosis). Certain factors arewell known in the art which may be used to identify those at risk ofdeveloping a bone deficit due to bone remodelling disorders likeosteoporosis. Important factors include low bone mass, family history,life style, estrogen or androgen deficiency and negative calciumbalance. Postmenopausal women are particularly at risk of developingosteoporosis. Hereinafter, references to treatment of bone diseases areintended to include management and/or prophylaxis except where thecontext demands otherwise.

The methods described herein can also be used to enhance bone formationin conditions where a bone deficit is caused by factors other than boneremodeling disorders. Such bone deficits include fractures, bone trauma,conditions associated with post-traumatic bone surgery, post-prostheticjoint surgery, post plastic bone surgery, post dental surgery, bonechemotherapy, post dental surgery and bone radiotherapy. Fracturesinclude all types of microscopic and macroscopic fractures. Examples offractures includes avulsion fracture, comminuted fracture, transversefracture, oblique fracture, spiral fracture, segmental fracture,displaced fracture, impacted fracture, greenstick fracture, torusfracture, fatigue fracture, intraarticular fracture (epiphysealfracture), closed fracture (simple fracture), open fracture (compoundfracture) and occult fracture.

As previously mentioned, a wide variety of bone diseases may be treatedin accordance with the present invention, for example all those bonediseases connected with the bone-remodeling cycle. Examples of suchdiseases include all forms of osteoporosis, osteomalacia, rickets andPaget's disease. Osteoporosis, especially of the post-menopausal, maleand steroid-induced types, is of particular note. In addition, thecompounds can be used as antiresorption agents generally, as bonepromotion agents and as anabolic bone agents. Such uses form anotheraspect of the present invention.

In many bone remodeling disorders, including osteoporosis, the bonedeficit may be attributed to excess bone resorption by differentiatedosteoclasts. The methods and compositions of the invention may beemployed to inhibit osteoclast differentiation, thus inhibiting boneresorption.

If desired, the lanthanum compound may be administered simultaneously orsequentially with other active ingredients. These active ingredientsmay, for example include other medicaments or compositions capable ofinteracting with the bone remodeling cycle and/or which are of use infracture repair. Such medicaments or compositions may, for example, bethose of use in the treatment of osteoarthritis or osteoporosis.

Bone enhancing agents, known in the art to increase bone formation, bonedensity or bone mineralisation, or to prevent bone resorption may beused in the methods and pharmaceutical compositions of the invention.Suitable bone enhancing agents include natural or synthetic hormones,such as estrogens, androgens, calcitonin, prostaglandins andparathormone; growth factors, such as platelet-derived growth factor,insulin-like growth factor, transforming growth factor, epidermal growthfactor, connective tissue growth factor and fibroblast growth factor;vitamins, particularly vitamin D; minerals, such as calcium, aluminum,strontium and fluoride; statin drugs, including pravastatin,fluvastatin, simvastatin, lovastatin and atorvastatin; agonsists orantagonsist of receptors on the surface of osteoblasts and osteoclasts,including parathormone receptors, estrogen receptors and prostaglandinreceptors; bisphosphonate and anabolic bone agents.

V. Cell-Based Assay

High throughput screening is a recent technology that has been developedprimarily within the pharmaceutical industry. It has emerged in responseto the profusion of new biological targets and the need of thepharmaceutical industry to generate novel drugs rapidly in a changedcommercial environment. Its development has been aided by the inventionof new instrumentation, by new assay procedures, and by the availabilityof databases that allow huge numbers of data points to be managedeffectively. High throughput screening combined with combinatorialchemistry, rational design, and automation of laboratory procedures hasled to a significantly accelerated drug discovery process compared tothe traditional one-compound-at-a-time approach. Screens may beperformed manually, however robotic screening of the compound librariesis preferred as a time- and labor-saving device.

One critical aspect of the drug discovery process is the identificationof potent lead compounds. A purely random selection of compounds fortesting is unlikely to yield many active compounds against a givenreceptor. Typically, pharmaceutical companies screen 100,000 or morecompounds per screen to identify approximately 100 potential leadcompounds. On average, only one or two of these compounds actuallyproduce lead compound series. Therefore, companies have been assayinglarger and larger data sets in the search for useful compounds. Compoundaccessibility then becomes an issue: historical compound collections arelimited in size and availability. In contrast, large combinatorialchemistry libraries can be synthesized on demand, but at significanttechnical difficulty and cost. As the library sizes expand, thedifficulty becomes selecting the desired compounds from these very largecombinatorial libraries. When literally hundreds of thousands ofcompounds are screened, it makes characterizing the candidate leadcompounds an expensive and time-consuming process, particularly whenmany of the “hits” turn out to be false positives. The multi-stepapproach to the drug discovery process described here provides asolution to many of these problems.

A high throughput bioassay to identify modulators that are selective forNR2C- or NR2D-containing receptors is also disclosed. High throughputscreening typically involves lead generation, followed by leadoptimization. NR2C/D-containing recombinant NMDA receptors show littledesensitization and are Ca⁺² permeable—two properties that renders themamenable to optical assays that measure agonist-induced Ca⁺²accumulation in mammalian cells using multi-well formats.

The assay involves using a cell line that expresses the NR1 subunittogether with either NR2C or NR2D. These cell lines can be prepared bytransfecting a cell line with an appropriate vector that includes theDNA encoding the NR2C or NR2D receptors. One suitable cell line is BHK-1(Syrian hamster kidney BHK-21 is a subclone (clone 13) of the parentalline established from the kidneys of five unsexed, one-day-old hamstersin 1961).

The NR2D receptor cDNA has also been cloned, for example, in 293T cells(Glover et al., “Interaction of the N-Methyl-D-Aspartic Acid ReceptorNR2D Subunit with the c-Abl Tyrosine Kinase*,” J. Biol. Chem., Vol. 275,Issue 17, 12725-12729, Apr. 28, 2000). The cDNA for NR2D is alsodescribed in this reference.

An NR2D cDNA (clone designation pNR2D422) is also disclosed in Arvanian,et al., “Viral Delivery of NR2D Subunits Reduces Mg2+ Block of NMDAReceptor and Restores NT-3-Induced Potentiation of AMPA-KainateResponses in Maturing Rat Motoneurons,” J Neurophysiol 92: 2394-2404,2004.

The cDNA for the NR2C is described, for example, in Lin, Y. J., Bovetto,S, Carver, J. M., and Giordano, T., “Cloning of the cDNA for the humanNMDA receptor NR2C subunit and its expression in the central nervoussystem and periphery, Molecular Brain Research, 1996, vol. 43, no 1-2,pp. 57-64 (41 ref.). Lin et al. describe several overlapping cDNA clonescontaining 3995 nucleotides of the human 2C NMDA receptor subunit (NR2C)that were isolated from human hippocampal and cerebellar cDNA libraries.The predicted protein sequence is 1233 amino acids long. Lin et al.noted that readily detectable levels of NR2C are present in thehippocampus, amygdala, caudate nucleus, corpus callosum, subthalamicnuclei and thalamus, as well as the heart, skeletal muscle and pancreas,demonstrating a widespread expression pattern of the NR2C gene, both inthe CNS and in the periphery.

In one embodiment, the high throughput bioassay usescommercially-available BHK-21 cell lines expressing NR1 under control ofthe Tet-On system (Clontech) (Hansen et al (2008), and whichconstitutively express either NR2C or NR2D. FIG. 4A illustrates vectordesign for the NR2D cell line. A similar strategy can be used for theNR2C cell line, except that the NR2C cDNA is used in place of NR2D cDNA.

Stable expression of NMDA receptor subunits is cytotoxic. To avoid thistoxicity, the culture media can be supplemented with NMDA receptorantagonists, for example, DL-APV and 7Cl-kynurenate. Functional NR1expression can be induced by doxycyclin before the assay.

Fura-2 Ca⁺² imaging of the functional response of the NR1/NR2D cell linecan be used to produce a glutamate EC₅₀ value, which can be compared tothat measured from two-electrode voltage-clamp assay. If these valuesare comparable, this suggests that the cell line faithfully reproducesNR1/NR2D properties.

A cell line, such as a BHK cell line which expresses a low affinityglutamate transporter system (K_(m) −40 μM) should help keep glutamateconcentration low, and reduce cytotoxicity due to NMDA receptorover-activation (Scott & Pateman, 1978; Arathoon & Telling, 1981).

BHK cells can be preferred, because they adhere tightly to the cultureplastic, allowing thorough washing of potentiators present duringculture without losing cells from the bottom of the dish. However, BHKcells can extrude a low level of glutamate through the reversal of thetransporter when glutamate is absent from the extracellular solution,such as during wash and dye loading. Because glutamate activatesNR2D-containing receptors with submicromolar EC₅₀ (<500 nM), even tensof nanomolar concentrations of glutamate (plus trace glycine) extrudedby BHK cells from time of washing through dye loading are sufficient toactivate NR1/NR2D receptors, injure cells, and compromise subsequentassays. This toxic activation also creates a high baseline Ca⁺² signal,which compromises the signal to noise ratio.

To circumvent this problem, one can remove cells from the incubator,wash out all antagonists, and subsequently add a competitive glycinesite antagonist, such as 7-Cl-kynurenate, during the dye loadingprotocol. Use of a relatively low affinity antagonist enhances cellhealth during dye loading and experimental setup by preventing continualNR1/NR2D receptor activation by low levels of glutamate extruded by BHKcells.

At the time of the assay, the competitive glycine site antagonist iseasily displaced by addition of an excess of glycine (for example,around 1 mM) together with glutamate (around 100 μM). The presence ofantagonist improves the reliability and the signal-to-noise ratio forthe assay.

One can vary plating density, culture time, induction time, DMSOcontent, agonist concentration, Ca⁺² concentration, fluorescent dyeloading conditions, recording duration, and other parameters to reducewell-to-well variability. Z′ values are a standard measure ofvariability for multi-well assays, with values above 0.5 considered agood indication that an assay is suitable for single well screening oftest compounds (Zhang et al. 1999).Z′=1−3′(SD _(signal) +SD _(baseline))/A _(signal) −A _(baseline)

We have carried out the assay, as shown in Example 8, and the assayalways yielded a favorable value for Z′ (0.4-0.8). Real time Ca⁺²signals can be recorded in multi-well plates, for example, 96 wellplates, using plate readers, for example, FlexStation II multi-modeplate readers.

The assay has been designed to identify modulators acting to alteragonist binding to NR2D-containing receptors by using supramaximalconcentrations of glutamate and glycine.

The assay can be validated using commercially available libraries, suchas the Lopac library (1200 compounds), which contain a number of knownNMDA receptor antagonists.

Test compounds can be added to each well, together with agonist, toyield a final well concentration of around 10 μM test compound in 0.9%DMSO. Compounds that alter the response of any well, compared toon-plate control wells, beyond 2.5-fold of the standard deviation(calculated from all wells on the plate) and by more than 40% of thecontrol response on a given plate, can be selected for secondaryscreening. This secondary screening can be performed, for example, usingtwo-electrode voltage-clamp recordings from Xenopus oocytes expressingrecombinant NR1/NR2D receptors.

In one embodiment, the library of candidate compounds is a focusedlibrary of candidate compounds, for lead optimization, based on thestructure of high affinity leads identified in a first lead generationassay.

The library of candidate compounds can be a combinatorial library of,for example drug-like molecules or a focused small molecule library.

The invention also provides compounds, including small molecules andpeptides, proteins, and genetic material, identified according to themethods described above, as well as methods of treating patients in needof a subtype specific NMDA modulator, which methods involveadministering the modulator to a patient in need of treatment thereof.

Any method known in the art for selecting and synthesizing smallmolecule libraries for screening is contemplated for use in thisinvention. Small molecules to be screened are advantageously collectedin the form of a combinatorial library. For example, libraries ofdrug-like small molecules, such as beta-turn mimetic libraries and thelike, may be purchased from for example ChemDiv, Pharmacopia orCombichem or synthesized and are described in Tietze and Lieb, Curr.Opin. Chem. Biol. 2:363-371, 1998; Carrell et al., Chem Biol. 2:171-183,1995; U.S. Pat. Nos. 5,880,972, 6,087,186 and 6,184,223, the disclosuresof which are hereby incorporated by reference.

Any of these libraries known in the art are suitable for screening, asare random libraries or individual compounds. In general, hydrophiliccompounds are preferred because they are more easily soluble, moreeasily synthesized, and more easily compounded. Compounds having anaverage molecular weight of about 500 often are most useful, however,compounds outside this range, or even far outside this range also may beused. Generally, compounds having c log P scores of about 5.0 arepreferred, however the methods are useful with all types of compounds.Simple filters like Lipinski's “rule of five” have predictive value andmay be used to improve the quality of leads discovered by this inventivestrategy by using only those small molecules which are bioavailable. SeeLipinski et al., Adv. Drug Delivery Rev. 23:3-25, 1997.

Combinatorial chemistry small molecule “libraries” can be screenedagainst drug targets. The idea is that diversity of chemical structuresincreases the chances of finding the needle in the 10²⁰⁰ possible smallorganic molecule haystack. These collections provide an excellent sourceof novel, readily available leads. For example, ChemDiv uses more than800 individual chemical cores, a unique Building Block Library, andproprietary chemistry in designing its Diversity Collections (smallmolecule libraries) to assemble 80,000-100,000 compounds a year.CombiLab lead library sets of 200-400 compounds also can be produced asa follow-up. In addition, ChemDiv's compounds are designed to ensuretheir similarity to drugs adjusted according to proprietary algorithmsof “drug-likeness definitions” (group similarity and advanced neural netapproaches), and a variety of intelligent instruments for ADME&T(Absorption, Distribution, Metabolism, Excretion and Toxicity)properties prediction, such as partition coefficient, solubility,dissociation coefficients, and acute toxicity.

Thus, focused synthesis of new small molecule libraries can provide avariety of compounds structurally related to the initial lead compoundwhich may be screened to choose optimal structures. Preferably, alibrary of compounds is selected that are predicted to be “drug-like”based on properties such as pKa, log P, size, hydrogen bonding andpolarity. The inventive multi-step approach which yields high affinitypeptides in the first step, and small molecules in a subsequent stepreduces the number of artificial hits by eliminating the lower affinitysmall molecules that would be selected and have to be assayed in anormal high throughput screening method. In addition, it focuses thesearch for molecules that can modulate the binding of a peptide themimics the G protein rather than screening for binding to any site onthe receptor. Other advantages of this technology are that it is simpleto implement, amenable to many different classes of receptors, andcapable of rapidly screening very large libraries of compounds.

Generally, it is convenient to test the libraries using a one well-onecompound approach to identify compounds which compete with the peptidefusion protein or high affinity peptide for binding to the receptor. Asingle compound per well can be used, at about 1 μM each or at anyconvenient concentration depending on the affinity of the receptor forthe compounds and the peptide against which they are being tested.Compounds may be pooled for testing, however this approach requiresdeconvolution. Compounds may be pooled in groups of about 2 to about 100compounds per well, or more, or about 10 to about 50 compounds per wellat about 10 nM each or at any convenient concentration depending on theaffinity of the receptor for the compounds being tested. Severaldifferent concentrations may be used if desired.

Peptides desirably are screened using a pooled approach because of thelayer members of peptides which are screened in the first instance.Peptides may be screened individually as well, but preferably arescreened in pools of about 10⁴-10¹² peptides per well or about 10⁸-10¹⁰peptide per well.

Preferably, the most strongly binding and effective compounds aresubjected to a subsequent lead optimization screening step.

Thorough evaluation of the selected compounds (either peptides or smallmolecules) for use as therapeutic agents may proceed according to anyknown method. Properties of the compounds, such as pK_(a), log P, size,hydrogen bonding and polarity are useful information. They may bereadily measured or calculated, for example from 2D connection tables,if not already known prior to identification by the inventive method asa useful compound. Association/dissociation rate constants may bedetermined by appropriate binding experiments. Parameters such asabsorption and toxicity also may be measured, as well as in vivoconfirmation of biological activity. The screen may be optimized forsmall molecules according to methods known in the art. Additionally, itis preferable to use a software system for presentation of data thatallows fast analysis of positives.

Many databases and computer software programs are available for use indrug design. For example, see Ghoshal et al., Pol. J. Pharmacol.48(4):359-377, 1996; Wendoloski et al., Pharmacol. Ther. 60(2):169-183,1993; and Huang et al., J. Comput. Aided Mol. Des. 11:21-78, 1997.Databases can be used to store and manipulate data on the compoundsobtained using the screen, and can compare the binding affinity againstthe NR2C and NR2D receptors, and/or other receptors, to determine theselectivity of the compounds for the desired receptor.

EXAMPLES

The following examples are provided to illustrate the present inventionand are not intended to limit the scope thereof. Those skilled in theart will readily understand that known variations of the conditions andprocesses of the following preparative procedures can be used tomanufacture the desired compounds. The materials required for theembodiments and the examples are known in the literature, readilycommercially available, or can be made by known methods from the knownstarting materials by those skilled in the art.

Example 1: NMDA Receptor Activity of the Compounds of Formula A

FIG. 2A illustrates the subunit-selectivity with which the compounds ofFormula A, including a specific compound herein referred to as DIQ-1180,acts on recombinant NMDA receptors. The structure of DIQ-1180 is shownbelow.

The EC₅₀ value for potentiation of NR2C-containing receptors was 11 μM(maximal potentiation 181%); the EC₅₀ value at NR2D-containing receptorswas 13 μM 162%; several halogenated analogues are ˜3-fold more potentwith EC₅₀ values under 3 μM (not shown). The Hill slope at bothreceptors for all analogues was greater than 1, consistent with acooperative effect of binding at two sites. Potentiation was reversible,repeatable, and did not lead to run down or run up of the response (datanot shown). The potentiating actions of DIQ-1180 werevoltage-independent (n=7) and pH-independent, suggesting thatpotentiation did not reflect relief of tonic proton inhibition(n=10-16), as has been proposed for spermine potentiation ofNR2B-containing receptors (Traynelis et al 1995). Furthermore, at peakpotentiation there is no shift in the EC₅₀ for activation of NR1/NR2Dreceptors by glutamate (EC₅₀ was 0.39 and 0.43 μM in the absence andpresence of DIQ-1180; n=6,6) or glycine, which had an EC₅₀ of 0.20 μMboth in the absence (n=5) and presence (n=8) of DIQ-1180.

These data suggest that potentiator binding is not allostericallycoupled to agonist binding. Analysis of glutamate/glycine-activatedunitary currents in an excised outside-out patch that contain at leasttwo NR1/NR2D channels suggest that DIQ-1180 can increase the total openprobability (nPo) from 0.083 to 0.207 (FIG. 3B). This reflected anincrease to 178% of control in opening frequency, which was reversed bywashout of compound DIQ-1180 (not shown). Our initial studies varyingthe compound structure confirm that there are strict requirements forthe position and type of substituent on the isoquinoline backbone (seeTable 3).

NR2C/D Expression in Hippocampal and Subthalamic Neurons

We tested compound-1180 for activity at native NMDA receptor responsesin neurons from basal ganglia slices. We focused on neurons that havebeen suggested to express NR2D. We performed patch clamp recording fromslices of subthalamic nuclei in our lab. Preliminary data shows thatDIQ-1180 (30 μM) potentiates the current response to pressure-appliedNMDA (n=2; FIG. 3) in slices bathed in tetrodotoxin (1 μM) to eliminatesynaptic activity.

These data are consistent with the idea derived from anatomical datasuggesting that subthalamic neurons likely express NMDA receptors thatcontain functional NR2D subunit (Standaert et al 1994; Dunah et al 1998,2003).

Data on additional potentiators is shown below in the following tables:

1180 P COMPOUNDS 2A 2B 2C 2D IC50 IC50 IC50 IC50 # Structure (μM) (μM)(μM) Max (μM) Max 1180

 81% at 100 μM  67% at 100 μM  12 145  11 156 1369

 94% at 100 μM  84% at 100 μM  7 184  7 169 1390

115% at 100 μM  82% at 100 μM  3 215  3 205 1391

119% at 100 μM  74% at 100 μM  1 195  2 188 1426

 88% at 100 μM  86% at 100 μM  2 171  2 204 1425

 74% at 100 μM  94% at 100 μM  5 208  5 172 1392

104% at 100 μM  59% at 100 μM  6 181  12 179 1368

105% at 100 μM  78% at 100 μM  4 135  5 114 1409

 76% at 100 μM  82% at 100 μM 1263

105% at 100 μM  97% at 100 μM 1371

 73% at 100 μM  70% at 100 μM  89% at 100 μM 100% at 100 μM 1408

 95% at 100 μM  88% at 100 μM 1364

 75% at 100 μM  98% at 100 μM  93% at 100 μM 1393

120% at 100 μM  80% at 100 μM  96% at 100 μM  94% at 100 μM 1370

 77% at 100 μM  78% at 100 μM  83% at 100 μM  95% at 100 μM 1510

 87% at 100 μM  90% at 100 μM  87% at 100 μM 1484

100% at 100 μM  80% at 100 μM  76% at 100 μM  84% at 100 μM 1485

 75% at 100 μM  85% at 100 μM  82% at 100 μM  74% at 100 μM 1486

 78% at 100 μM  88% at 100 μM  9 131  6 112 1487

 89% at 100 μM  71% at 100 μM  69% at 100 μM  79% at 100 μM 1511

 99% at  10 μM  96% at  10 μM 1367

104% at 100 μM  93% at 100 μM 1444

 91% at 100 μM  68% at 100 μM 102% at 100 μM 111% at 100 μM 1410

 94% at 100 μM  89% at 100 μM 1366

 98% at 100 μM  97% at 100 μM  3 121 108% at 100 μM 1394

101% at 100 μM  61% at 100 μM  71% at 100 μM  68% at 100 μM 1438

114% at 100 μM  85% at 100 μM  77% at 100 μM  87% at 100 μM 1439

 99% at 100 μM  96% at 100 μM  84% at 100 μM  89% at 100 μM 1440

123% at 100 μM  90% at 100 μM  71% at 100 μM  82% at 100 μM 1407

 86% at 100 μM  88% at 100 μM 1411

 84% at 100 μM  66% at 100 μM 1412

 93% at 100 μM  79% at 100 μM 1413

102% at 100 μM  74% at 100 μM 1414

 99% at 100 μM  93% at 100 μM 1415

 99% at 100 μM  87% at 100 μM 1416

 96% at 100 μM  87% at 100 μM

No compounds potentiated homomeric GluR1 AMPA receptor responses. Whenno inhibition or potentiation is given, the percent effect at themaximum tested concentration is given.

In the table above, potency is expressed as fitted EC₅₀ value to averagecomposite concentration-effect data constructed from current responsesrecorded under two electrode voltage clamp from Xenopus laevis oocytesexpressing either NR1/NR2A, B, C, or D, GluR1, or GluR6. No effect meansless than 20% change in response amplitude at 30 μM of drug.

1180 S Compounds 2A 2B 2C 2D IC50 IC50 IC50 IC50 # Structure (μM) (μM)(μM) Max (μM) Max 1180-1

110% at 100 μM 96% at 30 μM 90% at 30 μM 1180-2

100% at 100 μM 101% at 100 μM 77% at 100 μM 81% at 100 μM 1180-3

126% at 100 μM 78% at 100 μM 81% at 100 μM 79% at 100 μM 1180-4

105% at 100 μM 69% at 100 μM 100% at 100 μM 89% at 100 μM 1180-5

98% at 100 μM 86% at 100 μM 83% at 100 μM 81% at 100 μM 1180-6

93% at 100 μM 97% at 100 μM 94% at 100 μM 88% at 100 μM 1180-7

94% at 100 μM 92% at 100 μM 75% at 100 μM 75% at 100 μM 1180-8

99% at 100 μM 89% at 100 μM 73% at 100 μM 84% at 100 μM 1180-9

87% at 100 μM 97% at 100 μM 109% at 100 μM 99% at 100 μM 1180-10

3 134 8 172 1180-11

84% at 30 μM 82% at 30 μM 31 431 111% at 30 μM 1180-12

97% at 30 μM 90% at 30 μM 2 133 109% at 30 μM 1180-13

112% at 100 μM 67% at 100 μM 97% at 100 μM 105% at 100 μM 1180-14

115% at 100 μM 128% at 100 μM 93% at 100 μM 103% at 100 μM 1180-15

128% at 30 μM 2.4 330 4 364 1180-16

50% at 100 μM 123% at 100 μM 13 267 16 297 1180-17

50% at 20 uM 90% at 100 μM 95% at 100 μM 83% at 100 μM 1180-18

136% at 100 μM 95% at 100 μM 6 226 7 206 1180-19

63% at 100 μM 100% at 100 μM 10 160 9 150 1180-20

92% at 100 μM 86% at 100 μM 11 253 18 271 1180-21

92% at 100 μM 72% at 100 μM 7 265 7 230 1180-22

95% at 100 μM 50% at 20 μM 79% at 100 μM 58% at 100 μM 1180-23

140% at 100 μM 61% at 100 μM 87% at 100 μM 78% at 100 μM 1180-24

100% at 100 μM 96% at 100 μM 79% at 100 μM 83% at 10 0μM 1180-25

96% at 100 μM 76% at 100 μM 12 188 7 162 1180-26

93% at 100 μM 75% at 100 μM 0.8 196 1.0 185 1180-27

107% at 100 μM 77% at 100 μM 1 192 1.0 168 1180-28

71% at 100 μM 95% at 100 μM 78% at 100 μM 80% at 100 μM 1180-29

75% at 100 μM 58% at 100 μM 82% at 100 μM 1180-30

1180-31

95% at 30 μM 82% at 30 μM 1.2 190 1.5 172 1180-32

1180-33

1180-34

1180-35

No compounds potentiated homomeric GluR1 AMPA receptor responses. Whenno inhibition or potentiation is given, the percent effect at themaximum tested concentration is given.

1357 S Compounds 2A 2B 2C 2D IC50 IC50 IC50 IC50 # Structure (μM) (μM)Max (μM) Max (μM) Max 1357

102% at 100 μM 102% at 100 μM 175 388 120 326 1418

99% at 100 μM 115% at 100 μM 33 187 35 195 1421

106% at 100 μM 100% at 100 μM 41 159 35 152 1399

100% at 100 μM 108% at 100 μM 77 301 26 261 1417

87% at 100 μM 102% at 100 μM 20 202 16 205 1482

97% at 100 μM 116% at 100 μM 19 127 16 170 1481

113% at 100 μM 119% at 100 μM 97% at 100 μM 21 124 1406

75% at 100 μM 37 145 45 341 44 351 1423

121% at 100 μM 18 224 26 323 25 370 1424

123% at 100 μM 19 225 42 434 43 475 1434

121% at 100 μM 11 199 16 224 18 273 1435

107% at 100 μM 98% at 100 μM 103% at 100 μM 103% at 100 μM 1428

105% at 100 μM 70% at 100 μM 80% at 100 μM 72% at 100 μM 1395

92% at 100 μM 94% at 100 μM 95% at 100 μM 77% at 100 μM 1420

118% at 100 μM 97% at 100 μM 94% at 100 μM 75% at 100 μM 1489

95% at 100 μM 78% at 100 μM 83% at 100 μM 76% at 100 μM 1488

98% at 100 μM 83% at 100 μM 82% at 100 μM 80% at 100 μM 1404

86% at 100 μM 68% at 100 μM 99% at 100 μM 77% at 100 μM 1419

93% at 100 μM 91% at 100 μM 97% at 100 μM 90% at 100 μM 1396

96% at 100 μM 97% at 100 μM 95% at 100 μM 81% at 100 μM 1441

81% at 100 μM 43  0 78% at 100 μM 86% at 100 μM 1400

80% at 100 μM 89% at 100 μM 96% at 100 μM 86% at 100 μM 1433

97% at 100 μM 98% at 100 μM 51 134 34 150 1398

89% at 100 μM 102% at 100 μM 87% at 100 μM 76% at 100 μM 1401

105% at 100 μM 87% at 100 μM 97% at 100 μM 86% at 100 μM 1479

94% at 100 μM 97% at 100 μM 102% at 100 μM 113% at 100 μM 1480

92% at 100 μM 83% at 100 μM 86% at 100 μM 85% at 100 μMNo compounds potentiated homomeric GluR1 AMPA receptor responses. Whenno inhibition or potentiation is given, the percent effect at themaximum tested concentration is given.

1357 P Compounds and 1343 and 1568 Compounds 2A 2B 2C 2D IC50 IC50 IC50IC50 # Structure (μM) (μM) Max (μM) Max (μM) Max 1343

115% at 100 μM 138 0 139 303 210 480 1344

78% at 100 μM 174 0 88% at 100 μM 97% at 100 μM 1397

98% at 100 μM 87% at 100 μM 1402

104% at 100 μM 80% at 100 μM 103% at 100 μM 93% at 100 μM 1403

88% at 100 μM 94% at 100 μM 104% at 100 μM 86% at 100 μM 1563

91% at 100 μM  24 140  39 424  68 614

In the tables shown herein, in those embodiments where the nitrogen atomin an amide or thioamide linkage is not shown attached to three atoms,an N—H linkage is intended.

Example 2: In Vitro Binding Studies for Secondary Effects

Compounds can be evaluated for binding to the human ether-a-go-gopotassium channel (hERG) expressed in HEK293 cells by displacement of³[H]-astemizole according to the methods by Finlayson et al. (K.Finlayson., L. Turnbull, C. T. January, J. Sharkey, J. S. Kelly;[³H]Dofetilide binding to HERG transfected membranes: a potential highthroughput preclinical screen. Eur. J. Pharmacol. 2001, 430, 147-148).Compounds can be incubated at 1 or 10 μM final concentration, induplicate, and the amount of displaced ³[H]-astemizole determined byliquid scintillation spectroscopy. In some cases, a seven concentration(each concentration in duplicate) displacement curve can be generated todetermine an IC₅₀. Binding to the rat alpha-1 adrenergic receptor in ratbrain membranes can be determined by displacement of ³[H]-prazosin (P.Greengrass and R. Bremner; Binding characteristics of ³H-prazosin to ratbrain a-adrenergic receptors. Eur. J. Pharmacol. 1979, 55: 323-326).Compounds can be incubated at 0.3 or 3 μM final concentration, induplicate, and the amount of displaced ³[H]-prazosin determined byliquid scintillation spectroscopy. Binding IC₅₀ values can be determinedfrom displacement curves (four-six concentrations, each concentration induplicate) fit by a non-linear, least squares, regression analysis usingMathIQ (ID Business Solutions Ltd., UK). The binding Ki's can bedetermined from the IC₅₀ according to the method of Cheng and Prusoff(Y. Cheng and W. H. Prusoff; Relationship between the inhibitionconstant (K1) and the concentration of inhibitor which causes 50 percentinhibition (IC₅₀) of an enzymatic reaction. Biochem. Pharmacol. 1973,22: 3099-3108).

Example 3: Metabolic Stability

Compounds can be incubated with pooled human (from at least 10 donors)or rat liver microsomes, 1.0 mg/ml microsomal protein, and 1 mM NADPH,in buffer at 37° C. in a shaking water bath according to the method ofClarke and Jeffrey (S. E. Clarke and P. Jeffrey; Utility of metabolicstability screening: comparison of in vitro and in vivo clearance.Xenobiotica 2001. 31: 591-598). At 60 min the samples can be extractedand analyzed for the presence of the parent compound by LC-MS/MS. Theparent material remaining in the sample at 60 min can be compared tothat at 0 min and expressed as a percentage. A control compound,testosterone, can be run in parallel.

Rats (n=3 per dose) can be administered compounds at doses of 1-4 mg/kgin a single bolus i.v. infusion (2 ml/kg body weight) via the tail veinformulated in 2% dimethyl acetamide/98% 2-hydroxy-propyl cyclodextrin(5%). Animals can be fasted overnight prior to dose administration andfood returned to the animals two hours after dosing. Following IVdosing, blood samples (ca 200 μL) can be collected into separate tubescontaining anticoagulant (K-EDTA) via the orbital plexus at varioustimes post administration. Plasma samples can be prepared immediatelyafter collection by centrifugation for ten minutes using a tabletopcentrifuge, and stored at −80° C. Brain tissue can be weighed,homogenized on ice in 50 mM phosphate buffer (2 ml per brain) and thehomogenate stored at −80° C. Plasma and brain homogenate samples can beextracted by the addition of 5 volumes of cold acetonitrile, mixed wellby vortexing and centrifuged at 4000 rpm for 15 minutes. The supernatantfractions can be analyzed by LC-MS/MS operating in multiple reactionmonitoring mode (MRM). The amount of parent compound in each sample canbe calculated by comparing the response of the analyte in the sample tothat of a standard curve.

Example 5: High Throughput Screening Assay

A high throughput bioassay was developed to identify modulators that areselective for NR2C- or NR2D-containing receptors. NR2C orNR2D-containing recombinant NMDA receptors show little desensitizationand are Ca⁺² permeable—two properties that renders them amenable tooptical assays that measure agonist-induced Ca⁺² accumulation inmammalian cells using multi-well formats.

The high throughput bioassay used a commercially-available BHK-21 cellline expressing NR1 under control of the Tet-On system (Clontech)(Hansen et (12008) to create two new cell lines that constitutivelyexpress either NR2C or NR2D. FIG. 4A illustrates vector design for theNR2D cell line. A similar strategy was employed for the NR2C cell line,except that the NR2C cDNA replaced the NR2D cDNA.

Stable expression of NMDA receptor subunits is cytotoxic. To avoid thistoxicity, the culture media was supplemented with NMDA receptorantagonists (200 μM DL-APV and 200 μM 7Cl-kynurenate), and functionalNR1 expression was induced by doxycyclin 48 hours prior to assay (FIG.5B). Fura-2 Ca⁺² imaging of the functional response of the NR1/NR2D cellline (FIG. 5C) produced a glutamate EC₅₀ value (340 nM) that was similarto that measured from two-electrode voltage-clamp assay (460 nM),suggesting this cell line faithfully reproduces NR1/NR2D properties. TheBHK cell line expresses a low affinity glutamate transporter system(K_(m) −40 μM) which should help keep glutamate concentration low andreduce cytotoxicity due to NMDA receptor over-activation (Scott &Pateman, 1978; Arathoon & Telling, 1981).

In addition, these cells adhere tightly to the culture plastic, allowingthorough washing of antagonists present during culture without losingcells from the bottom of the dish. However, BHK cells can extrude a lowlevel of glutamate through the reversal of the transporter whenglutamate is absent from the extracellular solution, such as during washand dye loading.

Because glutamate activates NR2D-containing receptors with submicromolarEC₅₀ (<500 nM), even tens of nanomolar concentrations of glutamate (plustrace glycine) extruded by BHK cells from time of washing through dyeloading are sufficient to activate NR1/NR2D receptors, injure cells, andcompromise subsequent assays. This toxic activation also creates a highbaseline Ca⁺² signal, which compromises the signal to noise ratio.

To circumvent this problem, we removed cells from the incubator, washedout all antagonists, and subsequently added the competitive glycine siteantagonist 7-Cl-kynurenate (30 μM) during the dye loading protocol. Thisinvolved adding a cell permeant Ca²⁺ sensitive dye for 10-30 minutesbefore experimentation. This relatively low affinity antagonist enhancescell health during dye loading and experimental setup by preventingcontinual NR1/NR2D receptor or NR1/NR2C receptor activation by lowlevels of glutamate extruded by BHK cells. At the time of the assay, 30μM of the competitive glycine site antagonist 7-Cl-kynurenate is easilydisplaced by addition of an excess of glycine (1 mM) together withglutamate (100 μM; FIG. 5A). The presence of antagonist improved thereliability and the signal-to-noise ratio for the assay.

In another embodiment, however, one could alternatively add acompetitive glutamate site antagonist and, when the assay is performed,the competitive glutamate site antagonist is displaced by adding anexcess of glutamate together with glycine to improve the reliability andthe signal-to-noise ratio for the assay.

We varied plating density, culture time, induction time, DMSO content,agonist concentration. Ca²⁺ concentration, fluorescent dye loadingconditions, recording duration, and other parameters to reducewell-to-well variability. Z′ values are a standard measure ofvariability for multi-well assays, with values above 0.5 considered agood indication that an assay is suitable for single well screening oftest compounds (Zhang et al. 1999).Z′=1−3′(SD _(signal) +SD _(baseline))/A _(signal) −A _(baseline)

Our assay always yielded a favorable value for Z′ (0.4-0.8). Real timeCa⁺² signals were recorded in 96 well plates using a pair of FlexStationII multi-mode plate readers. The assay was designed to identifynon-competitive modulators of NR2D-containing receptors by usingsupramaximal concentrations of glutamate and glycine.

We validated our assay using the commercially available Lopac library(1200 compounds) and our own focused library (˜500 biarylnitrogen-containing compounds with ring systems separated by a defineddistance); these two libraries contained a number of known NMDA receptorantagonists in addition to several unpublished NMDA receptorpotentiators that we had previously identified. Test compounds wereadded to each well together with agonist to yield a final wellconcentration of 10 μM test compound in 0.9% DMSO.

Compounds that altered the response of any well compared to on-platecontrol wells beyond 2.5-fold of the standard deviation (calculated fromall wells on the plate) and by more than 40% of the control response ona given plate were selected for secondary screening using two-electrodevoltage-clamp recordings from Xenopus oocytes expressing recombinantNR1/NR2D receptors.

An NR1/NR2C expressing cell line was made using similar methods,optimized for single well screening, and tested for sensitivity to knownNMDA antagonists. The results obtained confirmed that the NR1/NR2C cellline was also well-suited for high-throughput screening.

Having hereby disclosed the subject matter of the present invention, itshould be apparent that many modifications, substitutions, andvariations of the present invention are possible in light thereof. It isto be understood that the present invention can be practiced other thanas specifically described. Such modifications, substitutions andvariations are intended to be within the scope of the presentapplication.

The invention claimed is:
 1. A pharmaceutical composition comprising acompound of the following formula:

or pharmaceutically acceptable salts or esters thereof wherein, R₁, R₃,and R₄ are selected independently from H, OMe, OH, SH, SMe, Cl, F, I,Br, C₁₋₆ alkyl, C₆₋₁₀ aryl, alkylaryl, and arylalkyl; R₂, is hydrogen;R₅ is a meta or para substituent and are selected independently fromOMe, OH, SH, SMe, CI, F, I, Br, —C(CH₂)OMe, C(O)OMe, ethyl, C₆₋₁₀ aryl,alkylaryl, and arylalkyl; R₆ is selected independently from H, OMe, OH,SH, SMe, I, Br, C₁₋₆ alkyl, C₆₋₁₀ aryl, alkylaryl, and arylalkyl; X isO; and Y is selected from S and O.
 2. A pharmaceutical compositioncomprising a compound of the following formula:

or pharmaceutically acceptable salts or esters thereof wherein, R₁, R₃and R₄, are selected independently from H, OMe, OH, SH, SMe, Cl, F, I,Br, C₁₋₆ alkyl, C₆₋₁₀ aryl, alkylaryl, and arylalkyl; R₂ is hydrogen; R₅is a meta or para substituent and are selected independently from OMe,OH, SH, SMe, CI, F, I, Br, —C(CH₂)OMe, C(O)OMe, ethyl, C₆₋₁₀ aryl,alkylaryl, and arylalkyl; R₆ is meta substituted OH, CF₃, OMe, Br, or I;X is O; and Y is selected from S and O.
 3. The pharmaceuticalcomposition of claim 1, wherein R₃ is methoxy.
 4. The pharmaceuticalcomposition of claim 2, wherein R₃ is methoxy.